Method for diagnosing non-small cell lung cancer

ABSTRACT

Disclosed are methods for detecting non-small cell lung cancer (NSCLC) using differentially expressed genes KIF11, GHSR1b, NTSR1, and FOXM1. Also disclosed are methods of identifying compounds for treating and preventing NSCLC, based on the interaction between KOC1 and KIF11, or NMU and GHSR1b or NTSR1.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Ser. No. 12/700,669,filed Feb. 4, 2010, which claims priority to U.S. Ser. No. 10/593,842,filed Jul. 10, 2007, now U.S. Pat. No. 7,700,573, which is a U.S.National Phase of International Application No. PCT/JP2005/005613, filedMar. 18, 2005, which claims priority to U.S. Ser. No. 60/555,789 filedMar. 23, 2004, all of which are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to the field of biological science, morespecifically to the field of cancer therapy and diagnosis. Inparticular, the invention relates to methods of diagnosing non-smallcell lung cancers using genes, KIF11, GHSR1b, NTSR1, and FOXM1, thatshow elevated expression in such cancerous cells.

BACKGROUND OF THE INVENTION

Lung cancer is one of the most commonly fatal human tumors. Many geneticalterations associated with the development and progression of lungcancer have been reported. Although genetic changes can aid prognosticefforts and predictions of metastatic risk or response to certaintreatments, information about a single or a limited number of molecularmarkers generally fails to provide satisfactory results for clinicaldiagnosis of non-small cell lung cancer (NSCLC) (Mitsudomi et al., ClinCancer Res 6: 4055-63 (2000); Niklinski et al., Lung Cancer. 34 Suppl 2:S53-8 (2001); Watine, BMJ 320: 379-80 (2000)). NSCLC is by far the mostcommon form, accounting for nearly 80% of lung tumors (Society, A.C.Cancer Facts and Figures 2001 (2001)). The overall 10-year survival rateremains as low as 10% despite recent advances in multi-modality therapy,because the majority of NSCLCs are not diagnosed until advanced stages(Fry, W. A. et al., Cancer 86: 1867-76 (1999)). Although chemotherapyregimens based on platinum are considered the reference standards fortreatment of NSCLC, those drugs are able to extend survival of patientswith advanced NSCLC only about six weeks (Non-small Cell Lung CancerCollaborative Group, BMJ. 311: 899-909 (1995)). Numerous targetedtherapies are being investigated for this disease, including tyrosinekinase inhibitors, but so far promising results have been achieved inonly a limited number of patients and some recipients suffer severeadverse reactions (Kris M. N. R., Herbst R. S. Proc. Am. Soc. Clin.Oncol. 21: 292a(A1166) (2002)).

Many genetic alterations associated with development and progression oflung cancer have been reported, but the precise molecular mechanismsremain unclear (Sozzi, G. Eur. J. Cancer 37: 63-73 (2001)). Over thelast decade newly developed cytotoxic agents including paclitaxel,docetaxel, gemcitabine, and vinorelbine have emerged to offer multipletherapeutic choices for patients with advanced NSCLC; however, each ofthe new regimens can provide only modest survival benefits compared withcisplatin-based therapies (Schiller, J. H. et al., N. Engl. J. Med. 346:92-98 (2002); Kelly, K. et al., J. Clin. Oncol. 19: 3210-3218 (2001)).Hence, new therapeutic strategies, such as development ofmolecular-targeted agents, are eagerly awaited by clinicians.

Systematic analysis of expression levels of thousands of genes on cDNAmicroarrays is an effective approach to identifying unknown moleculesinvolved in pathways of carcinogenesis (Kikuchi, T. et al., Oncogene 22:2192-2205 (2003); Kakiuchi, S. et al., Mol. Cancer Res. 1: 485-499(2003); Zembutsu, H. et al., Int. J. Oncol. 23: 29-39 (2003); Suzuki, C.et al., Cancer Res. 63: 7038-7041 (2003)) and can reveal candidatetargets for development of novel anti-cancer drugs and tumor markers. Toisolate novel molecular targets for diagnosis, treatment and preventionof NSCLC, pure populations of tumor cells were prepared from 37 cancertissues by laser-capture microdissection and genome-wide expressionprofiles of NSCLC cells were analyzed on a cDNA microarray containing23,040 genes (Kikuchi, T. et al., Oncogene 22: 2192-2205 (2003)). In thecourse of those experiments, KOC1 (GenBank Accession No. NM_(—)006547)and neuromedin U (NMU; GenBank Accession No. NM_(—)006681) wereidentified as genes that were frequently over-expressed in lung tumorsand indispensable for growth of NSCLC cells.

Cell-to-cell communication is a prerequisite for development andmaintenance of multicellular organisms. Several intercellularinformation-exchange systems such as chemical synapses, gap junctions,and plasmadesmata in plant cells have long been observed, but a newtransporting system involving a highly sensitive nanotubular structure,tunneling nanotubes (TNTs) between the cells, was only recently reportedin mammalian cells (Rustom, A. et al., Science 303, 1007-1010 (2004).Such a structure would facilitate the selective transfer of membranevesicles and organelles; therefore TNTs in mammalian somatic cells mightcontribute to a cell-to-cell transporting system(s) by carryingtranscription factors or ribonucleoparticles (RNPs), as in plants(Nakajima, K. et al., Nature 413, 307-311 (2001); Lucas, W. J. et al.,Nat. Rev. Mol. Cell Biol. 2, 849-857 (2001)). Some investigators havedocumented interactions between some RNA-binding proteins and motorproteins like kinesin and dynein within mammalian somatic cells, as wellas intercellular mRNA transport in mammalian germ cells (Brendza, R. P.et al., Science 289, 2120-2122 (2000); Chennathukuzhi, V. et al., Proc.Natl. Acad. Sci. USA 100, 15566-15571 (2003); Villace, P. et al.,Nucleic Acids Res. 32, 2411-2420 (2004); Morales, C. R. et al. Dev.Biol. 246, 480-494 (2002).). However, no report has emerged describingan intercellular mRNA transporting system in mammalian somatic cellsinvolving a complex of RNA-binding proteins and motor proteins. Thephenomenon of mRNA localization has been reported in oocytes anddeveloping embryos of Drosophila and Xenopus and in somatic cells suchas fibroblasts and neurons (King, M. L. et al., Bioessays 21: 546-557(1999); Mowry, K. L., Cote, C. A. FASEB J. 13: 435-445 (1999); Lasko, P.J. Cell Biol. 150: F51-56 (2000); Steward, O. Neuron 18: 9-12 (1997)).Beta actin (ACTB) mRNA is localized at the leading lamellae of chickenembryo fibroblasts (CEFs) (Lawrence, J. B., Singer, R. H. Cell 45:407-415 (1986)) and at the growth cone of developing neurons (Bassell,G. J. et al., J. Neurosci. 18: 251-265 (1998)). The localization of ACTBmRNA is dependent on the zipcode, a cis-acting element located in the 3′UTR of the mRNA (Kislauskis, E. H. et al., J. Cell Biol. 123: 165-172(1993)). The trans-acting factor, zipcode binding protein 1 (ZBP1), wasaffinity purified with the zipcode of ACTB mRNA (Ross, A. F. et al.,Mol. Cell Biol. 17, 2158-2165 (1997)). After the identification of ZBP1,additional homologues were identified in a wide range of organismsincluding Xenopus, Drosophila, human, and mouse (Mueller-Pillasch, F. etal., Oncogene 14: 2729-2733 (1997); Deshler, J. O. et al., Science 276:1128-1131 (1997); Doyle, G. A. et al., Nucleic Acids Res. 26: 5036-5044(1998)). ZBP1 family members are expressed in germ embryonic fibroblastsand in several types of cancer (Mueller-Pillasch, F. et al., Oncogene14: 2729-2733 (1997); Mueller, F. et al., Br. J. Cancer 88; 699-701(2003)). ZBP1-like proteins contain two RNA-recognition motifs (RRMs) atthe NH2-terminal part of the protein and four hnRNP K homology (KH)domains at the COOH-terminal end.

KOC1 (alias IGF-II mRNA-binding protein 3: IMP-3) is one of the IMPs(IMP-1, IMP-2, and IMP-3), which belong to the ZBP1 family members andexhibit multiple attachments to IGF-II leader 3 mRNA and thereciprocally imprinted H19 RNA (Mueller-Pillasch, F. et al., Oncogene14: 2729-2733 (1997)). Although KOC1 was initially reported to beover-expressed in pancreatic cancer (Mueller-Pillasch, F. et al.,Oncogene 14: 2729-2733 (1997); Mueller, F. et al., Br. J. Cancer 88:699-701 (2003)), its precise function in cancer cells or even in normalmammalian somatic cells remains unclear.

KOC1 is orthologous to the Xenopus Vg1 RNA-binding protein(Vg1RBP/Vera), which mediates the localization of Vg1 mRNA to thevegetal pole of the oocyte during oocyte maturation, and IMP-1 isorthologous to the ZBP1. IMP is mainly located at the cytoplasm and itscellular distribution ranges from a distinct concentration inperinuclear regions and lamellipodia to a completely delocalizedpattern. H19 RNA co-localized with IMP, and removal of the high-affinityattachment site led to delocalization of the truncated RNA (Runge, S. etal., J. Biol. Chem. 275: 29562-29569 (2000)), suggesting that IMPs areinvolved in cytoplasmic trafficking of RNA. IMP-1 was able to associatewith microtubles (Nielsen, F. C. et al., J. Cell Sci. 115: 2087-2097(2002); Havin, L. et al., Genes Dev. 12: 1593-1598 (1998)), and islikely to involve a motor protein such as kinesin, myosin, and dyenin.On the other hand, Oskar mRNA localization to the posterior polerequires Kinesin I (Palacios, I. M., St. Johnston D. Development 129:5473-5485 (2002); Brendza, R. P. et al., Science 289: 2120-2102 (2000)).

KIF11 (alias EG5) is a member of kinesin family, and plays a role inestablishing and/or determining the stability of specific microtublearrays that form during cell division. This role may encompass theability of KIF11 to influence the distribution of other proteincomponents associated with cell division (Whitehead, C. M., Rattner, J.B. J. Cell Sci. 111: 2551-2561 (1998); Mayer, T. U. et al., Science 286:971-974 (1999)).

NMU is a neuropeptide that was first isolated from porcine spinal cord.It has potent activity on smooth muscles (Minamino, N. et al., Biochem.Biophys. Res. Commun. 130: 1078-1085 (1985); Domin, J. et al., Biochem.Biophys. Res. Commun. 140: 1127-1134 (1986); Conlon, J. M. et al., J.Neurochem. 51: 988-991 (1988); Minamino, N. et al., Biochem. Biophys.Res. Commun. 156: 355-360 (1988); Domin, J. et al., J. Biol. Chem. 264:20881-20885 (1989), O'Harte, F. et al., Peptides 12: 809-812 (1991);Kage, R. et al., Regul. Pept. 33: 191-198 (1991); Austin, C. et al., J.Mol. Endocrinol. 12: 257-263 (1994); Fujii, R. et al., J. Biol. Chem.275: 21068-21074 (2000)), and in mammalian species NMU is distributedpredominantly in the gastrointestinal tract and central nervous system(Howard, A. D. et al., Nature 406: 70-74 (2000); Funes, S. et al.,Peptides 23: 1607-1615 (2002)). Peripheral activities of NMU includestimulation of smooth muscle, elevation of blood pressure, alternationof ion transport in the gut, and regulation of feeding (Minamino, N. etal., Biochem. Biophys. Res. Commun. 130: 1078-1085 (1985)); however, therole of NMU during carcinogenesis has not been addressed. Neuropeptidesfunction peripherally as paracrine and autocrine factors to regulatediverse physiologic processes and act as neurotransmitters orneuromodulators in the nervous system. In general, receptors thatmediate signaling by binding neuropeptides are members of thesuperfamily of G protein-coupled receptors (GPCRs) having seventransmembrane-spanning domains. Two known receptors for NMU, NMU1R andNMU2R, show a high degree of homology to other neuropeptide receptorssuch as GHSR and NTSR1, for which the corresponding known ligands areGhrelin (GHRL) and neurotensin (NTS), respectively. NMU1R (FM3/GPR66)and NMU2R (FM4) have seven predicted alpha-helical transmembrane domainscontaining highly conserved motifs, as do other members of the rhodopsinGPCR family (Fujii, R. et al., J. Biol. Chem. 275: 21068-21074 (2000);Howard, A. D. et al., Nature 406: 70-74 (2000); Funes, S. et al.,Peptides 23: 1607-1615 (2002)).

A C-terminal asparaginamide structure and the C-terminal hepatapeptidecore of NMU protein are essential for its contractile activity insmooth-muscle cells (Westfall, T. D. et al., J. Pharmacol. Exp. Ther.301: 987-992 (2002); Austin, C. J. Mol. Endocrinol. 14: 157-169 (1995)).Recent studies have contributed evidence that NMU acts at thehypothalamic level to inhibit food intake; therefore this protein mightbe a physiological regulator of feeding and body weight (Howard, A. D.et al., Nature 406: 70-74 (2000); Maggi, C. A. et al., Br. J. Pharmacol.99: 186-188 (1990); Wren, A. M. et al., Endocrinology 143: 227-234(2002); Ivanov, T. R. et al., Endocrinology 143: 3813-3821 (2002)).However, so far no reports have suggested involvement of NMUover-expression in carcinogenesis.

Studies designed to reveal mechanisms of carcinogenesis have alreadyfacilitated identification of molecular targets for anti-tumor agents.For example, inhibitors of farnesyltransferase (FTIs) which wereoriginally developed to inhibit the growth-signaling pathway related toRas, whose activation depends on posttranslational farnesylation, hasbeen effective in treating Ras-dependent tumors in animal models (He etal., Cell 99:335-45 (1999)). Clinical trials on human using acombination or anti-cancer drugs and anti-HER2 monoclonal antibody,trastuzumab, have been conducted to antagonize the proto-oncogenereceptor HER2/neu; and have been achieving improved clinical responseand overall survival of breast-cancer patients (Lin et al., Cancer Res.61:6345-9 (2001)). A tyrosine kinase inhibitor, STI-571, whichselectively inactivates bcr-abl fusion proteins, has been developed totreat chronic myelogenous leukemias wherein constitutive activation ofbcr-abl tyrosine kinase plays a crucial role in the transformation ofleukocytes. Agents of these kinds are designed to suppress oncogenicactivity of specific gene products (Fujita et al., Cancer Res. 61:7722-6(2001)). Therefore, gene products commonly up-regulated in cancerouscells may serve as potential targets for developing novel anti-canceragents.

It has been demonstrated that CD8+ cytotoxic T lymphocytes (CTLs)recognize epitope peptides derived from tumor-associated antigens (TAAs)presented on MHC Class I molecule, and lyse tumor cells. Since thediscovery of MAGE family as the first example of TAAs, many other TAAshave been discovered using immunological approaches (Boon, Int. J.Cancer 54: 177-80 (1993); Boon and van der Bruggen, J. Exp. Med. 183:725-9 (1996); van der Bruggen et al., Science 254: 1643-7 (1991);Brichard et al., J. Exp. Med. 178: 489-95 (1993); Kawakami et al., J.Exp. Med. 180: 347-52 (1994)). Some of the discovered TAAs are now inthe stage of clinical development as targets of immunotherapy. TAAsdiscovered so far include MAGE (van der Bruggen et al., Science 254:1643-7 (1991)), gp100 (Kawakami et al., J. Exp. Med. 180: 347-52(1994)), SART (Shichijo et al., J. Exp. Med. 187: 277-88 (1998)), andNY-ESO-1 (Chen et al., Proc. Natl. Acad. Sci. USA 94: 1914-8 (1997)). Onthe other hand, gene products which had been demonstrated to bespecifically over-expressed in tumor cells, have been shown to berecognized as targets inducing cellular immune responses. Such geneproducts include p53 (Umano et al., Brit. J. Cancer 84: 1052-7 (2001)),HER2/neu (Tanaka et al., Brit. J. Cancer 84: 94-9 (2001)), CEA (Nukayaet al., Int. J. Cancer 80: 92-7 (1999)), and so on.

In spite of significant progress in basic and clinical researchconcerning TAAs (Rosenbeg et al., Nature Med. 4: 321-7 (1998); Mukherjiet al., Proc. Natl. Acad. Sci. USA 92: 8078-82 (1995); Hu et al., CancerRes. 56: 2479-83 (1996)), only limited number of candidate TAAs for thetreatment of cancer are available. TAAs abundantly expressed in cancercells, and at the same time which expression is restricted to cancercells would be promising candidates as immunotherapeutic targets.Further, identification of new TAAs inducing potent and specificantitumor immune responses is expected to encourage clinical use ofpeptide vaccination strategy in various types of cancer (Boon and cander Bruggen, J. Exp. Med. 183: 725-9 (1996); van der Bruggen et al.,Science 254: 1643-7 (1991); Brichard et al., J. Exp. Med. 178: 489-95(1993); Kawakami et al., J. Exp. Med. 180: 347-52 (1994); Shichijo etal., J. Exp. Med. 187: 277-88 (1998); Chen et al., Proc. Natl. Acad.Sci. USA 94: 1914-8 (1997); Harris, J. Natl. Cancer Inst. 88: 1442-5(1996); Butterfield et al., Cancer Res. 59: 3134-42 (1999); Vissers etal., Cancer Res. 59: 5554-9 (1999); van der Burg et al., J Immunol 156:3308-14 (1996); Tanaka et al., Cancer Res. 57: 4465-8 (1997); Fujie etal., Int. J. Cancer 80: 169-72 (1999); Kikuchi et al., Int. J. Cancer81: 459-66 (1999); Oiso et al., Int. J. Cancer 81: 387-94 (1999)).

It has been repeatedly reported that peptide-stimulated peripheral bloodmononuclear cells (PBMCs) from certain healthy donors producesignificant levels of IFN-γ in response to the peptide, but rarely exertcytotoxicity against tumor cells in an HLA-A24 or -A0201 restrictedmanner in ⁵¹Cr-release assays (Kawano et al., Cancer Res. 60: 3550-8(2000); Nishizaka et al., Cancer Res. 60: 4830-7 (2000); Tamura et al.,Jpn. J. Cancer Res. 92: 762-7 (2001)). However, both of HLA-A24 andHLA-A0201 are one of the popular HLA alleles in Japanese, as well asCaucasian (Date et al., Tissue Antigens 47: 93-101 (1996); Kondo et al.,J. Immunol. 155: 4307-12 (1995); Kubo et al., J. Immunol. 152: 3913-24(1994); Imanishi et al., Proceeding of the eleventh InternationalHistocompatibility Workshop and Conference Oxford University Press,Oxford, 1065 (1992); Williams et al., Tissue Antigen 49: 129 (1997)).Thus, antigenic peptides of cancers presented by these HLAs may beespecially useful for the treatment of cancers among Japanese andCaucasian. Further, it is known that the induction of low-affinity CTLin vitro usually results from the use of peptide at a highconcentration, generating a high level of specific peptide/MHC complexeson antigen presenting cells (APCs), which will effectively activatethese CTL (Alexander-Miller et al., Proc. Natl. Acad. Sci. USA 93:4102-7 (1996)).

Although advances have been made in the development ofmolecular-targeting drugs for cancer therapy, the ranges of tumor typesthat respond as well as the effectiveness of the treatments are stillvery limited. Hence, it is urgent to develop new anti-cancer agents thattarget molecules highly specific to malignant cells and are likely tocause minimal or no adverse reactions. To achieve the goal moleculeswhose physiological mechanisms are well defined need to be identified. Apowerful strategy toward these ends would combine screening ofup-regulated genes in cancer cells on the basis of genetic informationobtained on cDNA microarrays with high-throughput screening of theireffect on cell growth, by inducing loss-of-function phenotypes with RNAisystems (Kikuchi, T. et al., Oncogene 22: 2192-2205 (2003)).

SUMMARY OF THE INVENTION

The present invention features a method of diagnosing or determining apredisposition to non-small cell lung cancer (NSCLC) in a subject bydetermining an expression level of a non-small cell lungcancer-associated gene that is selected from the group of KIF11, GHSR1b,NTSR1, and FOXM1 in a patient derived biological sample. An increase ofthe expression level of any of the genes compared to a normal controllevel of the genes indicates that the subject suffers from or is at riskof developing NSCLC.

The invention also provides methods of providing a prognosis of apatient diagnosed with NSCLC. In particular, the methods involvedetecting expression of KOC1, KIF11, or KOC1 in combination withexpression of KIF11.

A “normal control level” indicates an expression level of any of thegenes detected in a normal, healthy individual or in a population ofindividuals known not to be suffering from NSCLC. A control level is asingle expression pattern derived from a single reference population orfrom a plurality of expression patterns. In contrast to a “normalcontrol level”, the “control level” is an expression level of the genedetected in an individual or a population of individuals whosebackground of the disease state is known (i.e., cancerous ornon-cancerous). Thus, the control level may be determined base on theexpression level of the gene in a normal, healthy individual, in apopulation of individuals known not to be suffering from NSCLC, apatient of NSCLC or a population of the patients. The control levelcorresponding to the expression level of the gene in a patient ofnon-small cell lung cancer or a population of the patients is referredto as “NSCLC control level”. Furthermore, the control level can be adatabase of expression patterns from previously tested cells.

An increase in the expression level of any one of the genes of KIF11,GHSR1b, NTSR1, and FOXM1 detected in a test biological sample comparedto a normal control level indicates that the subject (from which thesample was obtained) suffers from NSCLC. Alternatively, the expressionlevel of any one or all of the genes in a biological sample may becompared to an NSCLC control level of the same gene(s).

Gene expression is increased or decreased 10%, 25%, 50% or more comparedto the control level. Alternatively, gene expression is increased ordecreased 1, 2, 5 or more fold compared to the control level. Expressionis determined by detecting hybridization, e.g., on a chip or an array,of an NSCLC gene probe to a gene transcript of a patient-derivedbiological sample. The patient-derived biological sample may be anysample derived from a subject, e.g., a patient known to or suspected ofhaving NSCLC. For example, the biological sample may be tissuecontaining sputum, blood, serum, plasma or lung cell.

The invention also provides a non-small cell lung cancer referenceexpression profile comprising a pattern of gene expression levels of twoor more genes selected from the group of KIF11, GHSR1b, NTSR1, andFOXM1.

The invention also provides a kit comprising two or more detectionreagents which detects the expression of one or more of genes selectedfrom the group of KIF11, GHSR1b, NTSR1, and FOXM1 (e.g., via detectingmRNA and polypeptide). Also provided is an array of polynucleotides thatbinds to one or more of the genes selected from the group of KIF11,GHSR1b, NTSR1, and FOXM1. The kits of the invention may also comprisereagents used to detect the expression of KIF11 and KOC1 to be used forthe prognosis of NSCLC. The invention also provides kits for thedetection of compounds that regulate RNA transporting activity. The kitsmay comprise a cell expressing a KIF11 polypeptide, or functionalequivalent, a KOC1 polypeptide, or functional equivalent, and RNA to betransported, and DCTN1. The kits of the invention may also be used toscreen for compounds for treating or preventing NSCLC. The kits maycomprise a KOC1 polypeptide, or functional equivalent, and an RNA thatis bound by the KOC1 polypeptide or functional equivalent.

The invention further provides methods of identifying compounds thatinhibit the expression level of an NSCLC-associated gene (KIF11, GHSR1b,NTSR1 or FOXM1) by contacting a test cell expressing an NSCLC-associatedgene with a test compound and determining the expression level of theNSCLC-associated gene. The test cell may be an NSCLC cell. A decrease ofthe expression level compared to a normal control level of the geneindicates that the test compound is an inhibitor of the expression orfunction of the NSCLC-associated gene. Therefore, if a compoundsuppresses the expression level of KIF11, GHSR1b, NTSR1 or FOXM1compared to a control level, the compound is expected to reduce asymptom of NSCLC.

Alternatively, the present invention provides a method of screening fora compound for treating or preventing NSCLC. The method includescontacting a polypeptide selected from the group of KIF11, GHSR1b,NTSR1, and FOXM1 with a test compound, and selecting the test compoundthat binds to or suppresses the biological activity of the polypeptide.The invention further provides a method of screening for a compound fortreating or preventing NSCLC, which includes the steps of contacting atest compound with a cell that expresses KIF11, GHSR1b, NTSR1 or FOXM1protein or introduced with a vector comprising the transcriptionalregulatory region of KIF11, GHSR1b, NTSR1 or FOXM1 gene upstream of areporter gene, and then selecting the test compound that reduces theexpression level of the KIF11, GHSR1b, NTSR1 or FOXM1 protein or proteinencoded by the reporter gene. According to these screening methods, thetest compound that suppresses the biological activity or the expressionlevel compared to a control level is expected to reduce a symptom ofNSCLC. Furthermore, the present invention provides a method of screeningfor a compound for treating or preventing NSCLC wherein the bindingbetween KIF11 and KOC1, or GHSR1b or NTSR1 and NMU is detected.Compounds that inhibit the binding between KIF11 and KOC1, or GHSR1b orNTSR1 and NMU are expected to reduce a symptom of NSCLC.

We detected a novel intra-cellular and inter-cellular RNA-transportingsystem in lung carcinomas, involving transactivation of KOC1 and KIF11.A complex of these two molecules in lung tumors was able to bind mRNAsencoding proteins known to function in intercellular adhesion,cancer-cell progression, and oncogenesis, and transport them toneighboring cells through ultrafine intercellular structures. Inparticular, evidence provided here shows that KOC1 binds to KIF11 at theRRM domain in the N-terminal region of KOC1. In addition, evidenceprovided here shows inhibition of their binding by dominant-negativeKOC1 mutants effectively suppressed growth of NSCLC cells in vitro. Forexample, KOC1 fragments (or nucleic acids encoding them) comprising theRRM domains of KOC1 can be used as dominant negative fragments tosuppress cell proliferation and thus treat cancer. Alternatively, theKOC1 fragment may comprise the ribonucleoprotein K-homologous (KH)domain.

The invention also provides methods of identifying polypeptides andother compounds that modulate RNA transport activity. For example, apolypeptide can be tested for RNA transporting activity by contactingthe polypeptide with a KIF11 polypeptide or a functional equivalentthereof with an RNA that can be transported by KIF11 under conditionssuitable for transportation of RNA. Alternatively, agents that modulateRNA transporting activity can be tested by contacting a test agent witha KIF11 polypeptide or a functional equivalent thereof with an RNA thatcan be transported by KIF11 under conditions suitable for transportationof RNA. Test agents useful for treating NSCLC by testing the agents forthe ability to inhibit binding between a KOC1 polypeptide, or afunctional equivalent, and an RNA that is bound by KOC1 or the complexof KOC1 and KIF11.

Immunohistochemical analysis of lung-cancer tissue microarraysdemonstrated that transactivation of KOC1 and KIF11 was significantlyassociated with poor prognosis of lung-cancer patients.

Methods for treating or preventing NSCLC and compositions to be used forsuch methods are also provided. Therapeutic methods include a method oftreating or preventing NSCLC in a subject by administering to thesubject a composition of an antisense, short interfering RNA (siRNA) ora ribozyme that reduce the expression of KIF11, GHSR1b, NTSR1 or FOXM1gene, or a composition comprising an antibody or fragment thereof thatbinds and suppresses the function of a polypeptide encoded by the gene.The compositions of the invention may also comprise a dominant negativeKOC1 mutant (or nucleic acids encoding it) comprising a KOC1 fragmentthat contains one or more RRM domains and/or KH domains of KOC1.

The invention also includes vaccines and vaccination methods. Forexample, a method of treating or preventing NSCLC in a subject iscarried out by administering to the subject a vaccine containing apolypeptide encoded by KIF11, GHSR1b, NTSR1 or FOXM1 gene, or animmunologically active fragment of the polypeptide. An immunologicallyactive fragment is a polypeptide that is shorter in length than thefull-length naturally-occurring protein and which induces an immuneresponse upon introduction into the body. For example, animmunologically active fragment includes a polypeptide of at least 8residues in length that stimulates an immune cell such as a T cell or aB cell in vivo. Immune cell stimulation can be measured by detectingcell proliferation, elaboration of cytokines (e.g., IL-2) or productionof antibody.

Other therapeutic methods include those wherein a compound selected bythe screening method of the present invention is administered.

Also included in the invention are double-stranded molecules thatcomprise a sense strand and an antisense strand. The sense strandcomprises a ribonucleotide sequence corresponding to a target sequencecomprised within the mRNA of a KIF11, GHSR1b, NTSR1 or FOXM1 gene, andthe antisense strand is a complementary sequence to the sense strand.Such double-stranded molecules of the present invention can be used assiRNAs against KIF11, GHSR1b, NTSR1 or FOXM1 gene. Furthermore, thepresent invention relates to vectors encoding the double-strandedmolecules of the present invention.

The present application also provides a composition for treating and/orpreventing NSCLC using any of the antisense polynucleotides or siRNAsagainst KIF11, GHSR1b, NTSR1 or FOXM1 gene, or an antibody that binds toa polypeptide encoded by KIF11, GHSR1b, NTSR1 or FOXM1 gene. Othercompositions include those that contain a compound selected by thescreening method of the present invention as an active ingredient.

It is to be understood that both the foregoing summary of the inventionand the following detailed description are of a preferred embodiment,and not restrictive of the invention or other alternate embodiments ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows photographs confirming the relationship between KOC1 andKIF11.

-   -   (a) depicts the result of co-immunoprecipitation of KOC1 and        KIF11 confirming the interaction between KOC1 and KIF11. A549        cells were transiently co-transfected with Flag-tagged KIF11 and        myc-tagged KOC1, immunoprecipitated with anti-Flag M2 agarose,        and subsequently immunoblotted with anti-myc antibody. In        contrast, using the same combination of vectors and cells, the        cells were immunoprecipitated with anti-myc agarose and        immunoblotted with anti-Flag M2 antibody. A band corresponding        to the immunoblotted protein was found only when both constructs        were co-transfected.

(b) depicts the result of immunocytochemical staining showing theco-localization of KOC1 and KIF11. COS-7 cells were transientlytransfected with FLAG-tagged KIF11 and myc-tagged KOC1, and theirco-localization was detected mainly in the cytoplasm using FITC-labeledanti-FLAG antibody and rhomamine-labeled anti-myc antibody.

(c) depicts the result of reciprocal co-immunoprecipitation ofendogenous KOC1 and KIF11 from extracts of lung-cancer cell lines A549and LC319. (upper panel) Western-blot analysis of both cell extractsimmunoprecipitated with anti-KOC1 antibodies, with KIF11 proteindetected in the immunoprecipitate. (lower panel) Western-blot ofextracts immunoprecipitated with anti-KIF11 antibodies, with KOC1protein detected in the immunoprecipitate.

FIG. 2 shows photographs confirming co-activation of KOC1 and KIF11 inlung tumors and normal tissues.

(a) depicts the result of QRT-PCR examining expression of KOC1 and KIF11in clinical samples of NSCLC and corresponding normal lung tissues.Y-axis indicates the relative expression rate of the two genes (KOC1 orKIF11/ACTS).

(b) depicts the result of QRT-PCR examining expression of KOC1 and KIF11among 20 lung-cancer cell lines.

(c) depicts the result of Northern-blot analysis detecting expression ofKOC1 and KIF11 in normal human tissues.

FIG. 3 shows photographs confirming the relationship between KOC1 andKIF11.

(a) shows schematic drawing of five KOC1 deletion mutants lacking eitheror both of the terminal regions, with N- and C-terminals tagged withFLAG and HA respectively. KH, ribonucleoprotein K-homologous domain.

(b) depicts the result of immunoprecipitation experiments foridentification of the region of KOC1 that binds to KIF11. The KOC1DEL4and KOC1DEL5 constructs, which lacked two RNA-recognition motifs, (RRM)did not retain any appreciable ability to interact with endogenousKIF11.

FIG. 4 shows photographs confirming the relationship between KOC1 andKOC1-associated mRNAs.

(a) depicts the result of Western blotting with immunoprecipitated KOC1deletion mutants and DIG-labeled RAB35 full length mRNA foridentification of the mRNA-binding region in KOC1.

(b) depicts the result of Northwestern with immunoprecipitated KOC1deletion mutants and DIG-labeled RAB35 full length mRNA foridentification of the mRNA-binding region in KOC1. The KOC1DEL3 andKOC1DEL5, did not bind to any of these mRNAs, and the KOC1DEL4, which isa construct with the four KH domains only, showed similar bindingaffinities for mRNAs to the KOC1DEL2, a construct without C-terminal twoKH domains.

(c) depicts the result of IP-RT-PCR for confirmation of IP-microarrayand the ability of various KOC1 deletion mutants transfected into A549cells to bind directly to representative eight endogenous mRNAs (CCT2,SBP2, SLC25A3, RAB35, PSMB7, GL, PKP4, and WINS1) among 55 candidategenes (see Table 2).

FIG. 5 shows photographs showing movement of KOC1-KIF11-mRNAribonuleoprotein complexes in living cultured mammalian cells.

(a) are photographs showing transport of the KOC1-KIF11 protein complex.Small particles that expressed fluorescent cyan (ECFP) KOC1 and yellow(EYFP) KIF11 proteins were co-localized, and transferred togetherbetween connected COS-7 cells through ultrafine intercellular structures(arrows).

(b) are photographs showing transport of KOC1-RAB35 mRNA RNP complexfrom one COS-7 cell that contains a high level of KOC1-RNP complex (cellA) to another cell with a lower level of the complex (stained simplywith CellTracker; cell B). Small particles of KOC1-RAB35 mRNA complex aswell as KOC1 particles were transferred from cell A to cell B throughultrafine intercellular structures (arrows).

FIG. 6 shows photographs showing localization of KOC1-KIF11-mRNAribonuleoprotein complexes.

(a) depicts the result of immunoprecipitation of cell extracts from A549and LC319 confirming of direct interaction between endogenous KIF11 andDCTN1 (upper and lower panels).

FIG. 7 shows photographs showing translation of KOC1-associated mRNAstransported into the recipient cells.

(a) are photographs showing translation of mRNA transported into therecipient cells monitored by in situ hybridization.

(b) are photographs showing protein synthesis based on transported mRNAin receiving cell. Constructs with full length RAB35 mRNA fused in frameto a myc tag sequence (upper panel). Co-localization of myc-tagged RAB35proteins in the cytoplasm of CellTracker-stained receiving cells usingimmunocytochemistry (lower panels).

(c) are photographs showing protein synthesis based on transported mRNAin receiving cell. Constructs with full length RAB35 mRNA fused in frameto a EGFP protein sequence.

(d) are photographs showing protein synthesis based on transported mRNAin receiving cell. Expression of EGFP-fused RAB35 proteins inCellTracker-positive receiving cells using time-lapse video microscopy.EGFP and related-DIC image were shown.

(e) are photographs showing that no significant difference in theprotein level of RAB35-EGFP fused-protein was found between COS-7 cellsthat were co-transfected with RAB35-EGFP and HA-tagged-KOC1 vectors, andthose with RAB35-EGFP and mock plasmid vectors. This indicates that KOC1is not likely to interfere with translation of RAB35-EGFP mRNA.

FIG. 8 shows the effect of KIF11 siRNAs on cells.

(a) depicts the inhibition on the growth of NSCLC cells by siRNAsagainst KIF11. The expression of KIF11 in response to specific siRNAs(si-KIF#1, #2, and #3) or control siRNAs (EGFP, LUC, SC) in A549 cells,was analyzed by semiquantitative RT-PCR.

(b) depicts the viability of A549 cells in response to si-KIF#1, #2, #3,EGFP, LUC, or SC, evaluated by triplicate MTT assays.

FIG. 9 shows the effect of KOC1 dominant-negative on cells.

(a) depicts the results of immunoprecipitation confirming interaction ofKOC1 deletion-mutant KOC1DEL3 with endogenous KIF11 in LC319 cells.

(b) depicts the results of immunoprecipitation confirming reduction ofthe complex formation between endogenous KOC1 and KIF11 in LC319 cellsover-expressing the RRM domains.

(c) depicts the viability of LC319 cells in response to dose-dependentdominant-negative effect of KOC1DEL3 evaluated by triplicate MTT assays.X-axis indicates dosage of KOC1DEL3 plasmid-DNA (μg) transfected intoLC319 cells in individual assays.

(d) depicts the results of immunoprecipitation detecting reduction ofthe complex formation between endogenous KOC1 and KIF11 in A549 cellsthat were transfected with the KOC1DEL2 construct.

(e) depicts the results of immunoprecipitation detecting interaction ofthe KOC1DEL2 with endogenous KIF11 in A549 cells.

(f) depicts the viability of A549 cells in response to dose-dependentdominant-negative effect of KOC1DEL2 evaluated by triplicate MTT assays.X-axis indicates dosage of KOC1DEL2 plasmid-DNA (μg) transfected intoA549 cells in individual assays.

FIG. 10 shows the effect of RAB35 siRNAs on cells.

(a) depicts the result of semi-quantitative RT-PCR analyzing mRNAknock-down effect in response to si-RAB35 or control siRNAs in A549cells.

(b) show results of MTT assays of A549 cells transfected with specificsiRNA or control plasmids (EGFP, Scramble, or Luciferase). Error barsrepresent the standard deviation of triplicate assays.

FIG. 11 shows association of KOC1 and KIF11 over-expression with worseoutcomes in NSCLC.

(a) depicts the results of immunohistochemical evaluation ofrepresentative samples from surgically-resected SCC tissues, usinganti-KOC1 (left) and anti-KIF11 (right) polyclonal antibodies on tissuemicroarrays (×200).

(b) depicts the results of Kaplan-Meier analysis of tumor-specificsurvival times according to KOC1 expression (left panel) and KIF11expression (right panel) on tissue microarrays.

FIG. 12 is schematic model for the mechanism of intracellular andcell-to-cell mRNA transport by KOC1-KIF11-DCTN1 complexes onmicrotubules. The KOC1 ribonucleoprotein complex, including KIF11motor-protein and DCTN1, transports KOC1-associated mRNAs through thestructure of microtubules within or between mammalian somatic cells.This model implies that proliferating cancer-cells may communicateactively by engaging this molecular complex in a system that transportsmRNAs critical for cancer growth or progression from one cell to anotherFIG. 8 shows the relationship between NMU and GHSR1b/NTSR1.

FIG. 13 (a) shows the result of semiquantitative RT-PCR analysisdepicting the expression of NMU, candidate receptors, and their knownligands detected in NSCLC cell lines.

(b) shows GHSR1b expression in normal human tissues.

(c) depicts the result of immunocytochemical staining using FITC-labeledanti-FLAG antibody showing the co-localization of NMU and GHSR1b/NTSR1on the cell surface of COS-7 cells that were transiently transfectedwith FLAG-tagged GHSR1b or NTSR1.

(d) depicts the interaction of NMU with GHSR1b/NTSR1. COS-7 cells weretransiently transfected with the same vectors, and binding ofrhodamine-labeled NMU-25 to the cell surface was detected by flowcytometry. As negative controls for these assays, three ligand/cellcombinations were prepared: 1) non-transfected COS-7 cells; 2)NMU-25-rhodamine vs non-transfected COS-7 cells; and 3) COS-7 cellstransfected only with GHSR1b or NTSR1.

(e) depicts the results of receptor-ligand binding assay using the LC319and PC-14 cells treated with NMU-25.

(f) depicts cAMP production of NMU-treated NSCLC cells.

FIG. 14 shows the effect of siRNAs on cells.

(a) depicts the inhibition on the growth of NSCLC cells by siRNAsagainst GHSR1b and NTSR1. Expression of GHSR or NTSR1 in response tospecific siRNAs (si-GHSR or si-NTSR1) or control siRNAs (EGFP, LUC, SCR)in A549 and LC319 cells were analyzed by semiquantitative RT-PCR.

(b) depicts the result of triplicate MTT assays evaluating viability ofA549 or LC319 cells in response to si-GHSR, NTSR1, EGFP, LUC, or SCR.

FIG. 15 shows validation of candidate downstream genes of NMU.

(a) depicts time-dependent reduction of NMU transcript by si-NMU.

(b) depicts the result of semiquantitative RT-PCR experiments of mRNAsfrom LC319 cells treated with si-NMU, with gene-specific primersconfirming time-dependent reduction of candidate downstream target geneexpression.

(c) depicts the result of semiquantitative RT-PCR using mRNAs from LC319cells incubated with NMU-25 or BSA (control) (100 μM) detectinginduction of FOXM1 as the candidate downstream target gene of NMU.

FIG. 16 shows the effect of FOXM1 siRNAs on cells.

(a) depicts inhibition of growth of NSCLC cells by siRNAs against FOXM1.Expression of FOXM1 in response to specific siRNAs (si-FOXM1) or controlsiRNAs (EGFP, LUC, SCR) in A549 cells, analyzed by semiquantitativeRT-PCR (upper panel). Viability of A549 cells, evaluated by triplicateMTT assays, in response to si-FOXM1, EGFP, LUC, or SCR (lower panel).

(b) depicts inhibition of growth of NSCLC cells by siRNAs against FOXM1.Expression of FOXM1 in response to specific siRNAs (si-FOXM1) or controlsiRNAs (EGFP, LUC, SCR) in LC319 cells, analyzed by semiquantitativeRT-PCR (upper panel). Viability of A549 cells, evaluated by triplicateMTT assays, in response to si-FOXM1, EGFP, LUC, or SCR (lower panel).

FIG. 17 is a schematic model for promotion of cancer cell growth andinvasion through the NMU-receptor interaction in the autocrineNMU-GHSR1b oncogenic signaling pathway. Binding of NMU to GHSR1b and/orNTSR1 leads to the activation of adenylate cyclase, accumulation ofintracellular cAMP and following activation of cAMP-dependent proteinkinase (PKA). The release of catalytic subunits of PKA (C) from theregulatory subunits (R) is resulting in the activation of downstreamFOXM1 gene and/or related target genes.

DETAILED DESCRIPTION OF THE INVENTION

The words “a”, “an” and “the” as used herein mean “at least one” unlessotherwise specifically indicated. The terms “protein” and “polypeptide”are used interchangeably. Furthermore, the terms “gene”,“polynucleotide”, and “nucleic acids” are used interchangeably unlessotherwise specifically indicated.

To investigate the mechanisms of lung carcinogenesis and identify genesthat might be useful as diagnostic markers or targets for development ofnew molecular therapies, genes specifically up-regulated in non-smallcell lung cancers (NSCLC) were searched by means of cDNA microarray.Through the analysis, a couple of candidate therapeutic target geneswere identified. Two genes, KH domain containing protein over-expressedin cancer (KOC1) and neuromedin U (NMU) were abundantly expressed inclinical NSCLC samples as well as NSCLC cell lines examined. However,their expression was hardly detectable in corresponding non-cancerouslung tissue. The growth of NSCLC cells that over-expressed endogenousNMU was significantly inhibited by anti-NMU antibody. Furthermore, thetreatment of NSCLC cells with siRNA against KOC1 and/or NMU suppressedthe expression of the gene and resulted in growth inhibition of theNSCLC cells. Furthermore, KOC1 was identified to bind to kinesin familymember 11 (KIF11) of the cancer cells, whereas NMU bound to theneuropeptide G protein-coupled receptors (GPCRs), growth hormonesecretagogue receptor 1b (GHSR1b) and neurotensin receptor 1 (NTSR1).NMU ligand-receptor system was identified to activate Homo sapiensforkhead box M1 (FOXM1). Interestingly, GHSR1b, NTSR1, FOXM1, and KIF11were all specifically over-expressed in NSCLC cells.

RNA binding protein KOC1 and microtubles motor protein KIF11 is requiredfor the localization of some kinds of mRNA needed in embryogenesis andcarcinogenesis (FIG. 12). As previously reported by the presentinventors, treatment of NSCLC cells with specific siRNA to reduceexpression of KOC1 resulted in growth suppression. In this study, KIF11was demonstrated to associate with KOC1 in NSCLC cells and to be thetarget for the growth-promoting effect of KOC1 in lung tumors. Thepresent inventors revealed that KOC1 not only co-localized with KIF11 inhuman normal tissues, NSCLCs, and cell lines, but also directlyinteracted with KIF11 in NSCLC cells in vitro, and that the treatment ofNSCLC cells with siRNAs for KIF11 reduced its expression and led togrowth suppression. The results show that KOC1-KIF11 signaling affectsgrowth of NSCLC cells. As shown below, dominant negative fragments ofKOC1 (e.g., those containing the RRM domains) can be used to inhibitproliferation of cancer cells. By expression analysis, increasedexpression of KOC1 and KIF11 was detected in the majority of NSCLCsamples, but not in normal lung tissues. Since most of the clinicalNSCLC samples used for the present analysis were at an early andoperable stage, KOC1 and KIF11 can be conveniently used as a biomarkerfor diagnosing early-stage lung cancer, in combination with fiberscopictransbronchial biopsy (TBB) or sputum cytology.

Therefore, KOC1 and KIF11 are essential for an oncogenic pathway inNSCLCs. The data reported here provide evidence for designing newanti-cancer drugs, specific for lung cancer, which target the KOC1-KIF11pathway. They also show that siRNAs can be used to treatchemotherapy-resistant, advanced lung cancers.

A significant increase in the sub-G1 fraction of NSCLC cells transfectedwith siRNA-NMU suggested that blocking the autocrine NMU-signalingpathway could induce apoptosis. The present inventors also found otherevidence supporting the significance of this pathway in carcinogenesis;e.g., addition of NMU into the medium promoted the growth of COS-7 cellsin a dose-dependent manner, and addition of anti-NMU antibody into theculture medium inhibited this NMU-enhanced cell growth, possibly byneutralizing NMU activity. Moreover, the growth of NSCLC cells thatendogenously over-expressed NMU was significantly inhibited by anti-NMUantibody. The expression of NMU also resulted in significant promotionof COS-7 cell invasion in in vitro assays. These results show that NMUis an important growth factor for NSCLC and is associated with cancercell invasion, functioning in an autocrine manner, and that screeningmolecules targeting the NMU-receptor growth-promoting pathway is auseful therapeutic approach for treating NSCLCs. By immunohistochemicalanalysis, increased expression of NMU protein was detected in themajority of NSCLC (SCC, ADC, LCC, and BAC) and SCLC samples, but not innormal lung tissues. Since NMU is a secreted protein and most of theclinical NSCLC samples used for the present analysis were at an earlyand operable stage, NMU can be conveniently used as a biomarker fordiagnosis of early-stage lung cancer, in combination with fiberscopictransbronchial biopsy (TBB), sputum cytology, or blood tests.

Two receptors, NMU1R (FM3/GPR66) and NMU2R (FM4) are known to interactwith NMU. The results presented here, however, indicated that these twoknown receptors were not the targets for the autocrine NMU-signalingpathway in NSCLCs; instead, GHSR1b and NTSR1 proved to be the targetsfor the growth-promoting effect of NMU in lung tumors. The presentinventors revealed that NMU-25 bound to these receptors on the cellsurface, and that treatment of NSCLC cells with siRNAs for GHSR1b orNTSR1 reduced expression of the receptors and led to apoptosis. Theresults show that NMU affects growth of NSCLC cells by acting throughGHSR1b and/or NTSR1 (FIG. 14). GHSR is a known receptor of Ghrelin(GHRL), a recently identified 28-amino-acid peptide capable ofstimulating release of pituitary growth hormone and appetite in humans(Lambert, P. D. et al., Proc. Natl. Acad. Sci. 98: 4652-4657 (2001);Petersenn, S. et al., Endocrinology 142: 2649-2659 (2001); Kim K. etal., Clin. Endocrinol. 54: 705-860 (2001); Kojima, M. et al., Nature402: 656-660 (1999)). Of the two transcripts known to be receptors forGHRL, GHSR1a and GHSR1b, over-expression of only GHSR1b was detected inNSCLC tissues and cell lines. Since GHRL was not expressed in the NSCLCsexamined, GHSR1b was suspected to have a growth-promoting function inlung tumors through binding to NMU, but not to GHRL.

NTSR1 is one of three receptors of neurotensin (NTS), a brain andgastrointestinal peptide that fulfills many central and peripheralfunctions (Heasley, L. E. Oncogene 20: 1563-1569 (2001)). NTS modulatestransmission of dopamine and secretion of pituitary hormones, and exertshypothermic and analgesic effects in the brain while it functions as aperipheral hormone in the digestive tract and cardiovascular system.Others have reported that NTS is produced and secreted in several humancancers, including small-cell lung cancers (SCLC) (Heasley, L. E.Oncogene 20: 1563-1569 (2001)). The expression of NTS was detected infour of the 15 NSCLC cell lines that were examined in the presentinvention (FIG. 13 a), but the expression pattern of NTS was notnecessarily concordant with that of NMU or NTSR1. Therefore NTS may,along with NMU, contribute to the growth of NSCLC through NTSR1 or otherreceptor(s) in a small subset of NSCLCs. In the present experiments themajority of the cancer cell lines and clinical NSCLCs that expressed NMUalso expressed GHSR1b and/or NTSR1, indicating that theseligand-receptor interactions were involved in a pathway that is centralto the growth-promoting activity of NMU in NSCLCs.

NMU signaling pathway affects the growth promotion of lung-cancer cellsby transactivating a set of downstream genes including FOXM1. FOXM1 wasknown to be over-expressed in several types of human cancers (Teh, M. T.et al., Cancer Res. 62, 4773-4780; van den Boom, J. et al., (2003). Am.J. Pathol. 163, 1033-1043; Kalinichenko, V. V. et al., (2004). Genes.Dev. 18, 830-850). The “forkhead” gene family, originally identified inDrosophila, comprises transcription factors with a conserved 100-aminoacid DNA-binding motif, and has been shown to play important roles inregulating the expression of genes involved in cell growth,proliferation, differentiation, longevity, and transformation.Cotransfection assays in the human hepatoma HepG2 cell line demonstratedthat FOXM1 protein stimulated expression of both the cyclin B1 (CCNB1)and cyclin D1 (CCND1) (Wang, X. et al., (2002). Proc. Nat. Acad. Sci.99, 16881-16886.), suggesting that these cyclin genes are direct FOXM1transcription targets and that FOXM1 controls the transcription networkof genes that are essential for cell division and exit from mitosis. Itshould be noted that we observed activation of CCNB1 in the majority ofa series of NSCLC and its good concordance of the expression to FOXM1(data not shown). The promotion of cell growth in NSCLC cells by NMUmight reflect transactivation of FOXM1, which would affect the functionof those molecular pathways in consequence. Therefore, NMU, two newlyrevealed receptors for this molecule, GHSR1b and NTSR1, and theirdownstream gene FOXM1 are involved in an autocrine growth-promotingpathway in NSCLCs. The data reported here provide the basis fordesigning new anti-cancer drugs, specific for lung cancer, that targetthe NMU-GHSR1b/NTSR1-FOXM1 pathway. They also show that siRNAs thatinterfere with this pathway can be used to treat chemotherapy-resistant,advanced lung cancers.

These data show that KOC1-KIF11 signaling pathway is frequentlyup-regulated in lung carcinogenesis, and that NMU an important autocrinegrowth factor for NSCLC, acting through GHSR1b and NTSR1 receptormolecules. Thus, selective suppression of components of these complexescan suppress the development and/or progression of lung carcinogenesisand targeting these pathways are conveniently used in therapeutic anddiagnostic strategies for the treatment of lung-cancer patients.

Diagnosing Non-Small Cell Lung Cancer (NSCLC)

By measuring the expression level of KIF11, GHSR1b, NTSR1 or FOXM1 genein a biological derived from a subject, the occurrence of NSCLC or apredisposition to develop NSCLC in the subject can be determined. Theinvention involves determining (e.g., measuring) the expression level ofat least one, and up to all of KIF11, GHSR1b, NTSR1, and FOXM1 gene inthe biological sample.

According to the present invention, a gene transcript ofNSCLC-associated gene, KIF11, GHSR1b, NTSR1 or FOXM1, is detected fordetermining the expression level of the gene. The expression level of agene can be detected by detecting the expression products of the gene,including both transcriptional and translational products, such as mRNAand proteins. Based on the sequence information provided by the GenBank™database entries for the known sequences, KIF11 (NM_(—)004523), GHSR1b(NM_(—)004122), NTSR1 (NM_(—)002531), and FOXM1 (No. NM_(—)202003) genescan be detected and measured using techniques well known to one ofordinary skill in the art. The nucleotide sequences of the KIF11,GHSR1b, NTSR1, and FOXM1 genes are described as SEQ ID NOs: 1, 3, 5, and106, respectively, and the amino acid sequences of the proteins encodedby the genes are described as SEQ ID NOs: 2, 4, 6, and 107.

For example, sequences within the sequence database entriescorresponding to KIF11, GHSR1b, NTSR1 or FOXM1 gene can be used toconstruct probes for detecting their mRNAs by, e.g., Northern blothybridization analysis. The hybridization of the probe to a genetranscript in a subject biological sample can be also carried out on aDNA array. The use of an array is preferred for detecting the expressionlevel of a plurality of the NSC genes (KIF11, GHSR1b, NTSR1, and FOXM1).As another example, the sequences can be used to construct primers forspecifically amplifying KIF11, GHSR1b, NTSR1 or FOXM1 gene in, e.g.,amplification-based detection methods such as reverse-transcriptionbased polymerase chain reaction (RT-PCR). Furthermore, the expressionlevel of KIF11, GHSR1b, NTSR1 or FOXM1 gene can be analyzed based on thequantity of the expressed proteins encoded by the gene. A method fordetermining the quantity of the expressed protein includes immunoassaymethods. Alternatively, the expression level of KIF11, GHSR1b, NTSR1 orFOXM1 gene can also be determined based on the biological activity ofthe expressed protein encoded by the gene. For example, a proteinencoded by KIF11 gene is known to bind to KOC1, and thus the expressionlevel of the gene can be detected by measuring the binding ability toKOC1 due to the expressed protein. Furthermore, KIF11 protein is knownto have a cell proliferating activity. Therefore, the expression levelof KIF11 gene can be determined using such cell proliferating activityas an index. On the other hand GHSR1b and NTSR1 proteins are known tobind to NMU, and also have a cell proliferating activity. Thus,similarly to KIF11, the expression levels of GHSR1b and NTSR1 genes canbe detected by measuring their binding ability to NMU or cellproliferating activity due to the expressed protein.

Any biological materials may be used as the biological sample fordetermining the expression level so long as any of the KIF11, GHSR1b,NTSR1, and FOXM1 genes can be detected in the sample and includes testcell populations (i.e., subject derived tissue sample). Preferably, thebiological sample comprises a lung cell (a cell obtained from the lung).Gene expression may also be measured in blood, serum or other bodilyfluids such as sputum. Furthermore, the test sample may be cellspurified from a tissue.

The subject diagnosed for NSCLC according to the method is preferably amammal and includes human, non-human primate, mouse, rat, dog, cat,horse and cow.

The expression level of one or more of KIF11, GHSR1b, NTSR1 or FOXM1gene in the biological sample is compared to the expression level(s) ofthe same genes in a reference sample. The reference sample includes oneor more cells with known parameters, i.e., cancerous or non-cancerous.The reference sample should be derived from a tissue type similar tothat of the test sample. Alternatively, the control expression level maybe determined based on a database of molecular information derived fromcells for which the assayed parameter or condition is known.

Whether or not a pattern of the gene expression levels in a biologicalsample indicates the presence of NSCLC depends upon the composition ofthe reference cell population. For example, when the reference cellpopulation is composed of non-cancerous cells, a similar gene expressionlevel in the test biological sample to that of the reference indicatesthat the test biological sample is non-cancerous. On the other hand,when the reference cell population is composed of cancerous cells, asimilar gene expression profile in the biological sample to that of thereference indicates that the test biological sample includes cancerouscells.

The test biological sample may be compared to multiple referencesamples. Each of the multiple reference samples may differ in the knownparameter. Thus, a test sample may be compared to a reference sampleknown to contain, e.g., NSCLC cells, and at the same time to a secondreference sample known to contain, e.g., non-NSCLC cells (normal cells).

According to the invention, the expression of one or more of theNSCLC-associated genes, KIF11, GHSR1b, NTSR1, and FOXM1, is determinedin the biological sample and compared to the normal control level of thesame gene. The phrase “normal control level” refers to an expressionprofile of KIF11, GHSR1b, NTSR1 or FOXM1 gene typically found in abiological sample derived from a population not suffering from NSCLC.The expression level of KIF11, GHSR1b, NTSR1 or FOXM1 gene in thebiological samples from a control and test subjects may be determined atthe same time or the normal control level may be determined by astatistical method based on the results obtained by analyzing theexpression level of the gene in samples previously collected from acontrol group. An increase of the expression level of KIF11, GHSR1b,NTSR1 or FOXM1 gene in the biological sample derived from a patientderived tissue sample indicates that the subject is suffering from or isat risk of developing NSCLC.

An expression level of KIF11, GHSR1b, NTSR1 or FOXM1 gene in a testbiological sample can be considered altered when the expression leveldiffers from that of the reference by more than 1.0, 1.5, 2.0, 5.0, 10.0or more fold. Alternatively, an expression level of KIF11, GHSR1b, NTSR1or FOXM1 gene in a test biological sample can be considered altered,when the expression level is increased or decreased to that of thereference at least 50%, 60%, 80%, 90% or more.

The difference in gene expression between the test sample and areference sample may be normalized to a control, e.g., housekeepinggene. For example, a control polynucleotide includes those whoseexpression levels are known not to differ between the cancerous andnon-cancerous cells. The expression levels of the control polynucleotidein the test and reference samples can be used to normalize theexpression levels detected for KIF11, GHSR1b, NTSR1 or FOXM1 gene. Thecontrol genes to be used in the present invention include β-actin,glyceraldehyde 3-phosphate dehydrogenase and ribosomal protein P1.

The differentially expressed KIF11, GHSR1b, NTSR1 or FOXM1 geneidentified herein also allow for monitoring the course of treatment ofNSCLC. In this method, a test biological sample is provided from asubject undergoing treatment for NSCLC. If desired, multiple testbiological samples are obtained from the subject at various time pointsbefore, during or after the treatment. The expression of one or more ofKIF11, GHSR1b, NTSR1 or FOXM1 gene in the sample is then determined andcompared to a reference sample with a known state of NSCLC that has notbeen exposed to the treatment.

If the reference sample contains no NSCLC cells, a similarity in theexpression level of KIF11, GHSR1b, NTSR1 or FOXM1 gene in the testbiological sample and the reference sample indicates the efficaciousnessof the treatment. However, a difference in the expression level ofKIF11, GHSR1b, NTSR1 or FOXM1 gene in the test and the reference samplesindicates a less favorable clinical outcome or prognosis. In particular,increased expression of KOC1, KIF11, or KOC1 in combination withincreased expression of KIF11 is significantly associated with poorprognosis.

The term “efficacious” refers that the treatment leads to a reduction inthe expression of a pathologically up-regulated gene (including thepresent indicator genes, KIF11, GHSR1b, NTSR1, and FOXM1), or a decreasein size, prevalence or metastatic potential of NSCLC in a subject. Whena treatment is applied prophylactically, “efficacious” means that thetreatment retards or prevents occurrence of NSCLC or alleviates aclinical symptom of NSCLC. The assessment of NSCLC can be made usingstandard clinical protocols. Furthermore, the efficaciousness of atreatment is determined in association with any known method fordiagnosing or treating NSCLC. For example, NSCLC is diagnosedhistopathologically or by identifying symptomatic anomalies such aschronic cough, hoarseness, coughing up blood, weight loss, loss ofappetite, shortness of breath, wheezing, repeated bouts of bronchitis orpneumonia and chest pain.

Moreover, the present method for diagnosing NSCLC may also be appliedfor assessing the prognosis of a patient with the cancer by comparingthe expression level of KIF11, KOC1, GHSR1b, NTSR1, FOXM1 gene, or acombination thereof (e.g., KOC1 and KIF11) in the patient-derivedbiological sample. Alternatively, the expression level of the gene(s) inthe biological sample may be measured over a spectrum of disease stagesto assess the prognosis of the patient.

An increase in the expression level of KIF11, KOC1, GHSR1b, NTSR1 orFOXM1 gene compared to a normal control level indicates less favorableprognosis. A similarity in the expression level of KIF11, KOC1, GHSR1b,NTSR1 or FOXM1 gene compared to a normal control level indicates a morefavorable prognosis of the patient. Preferably, the prognosis of asubject can be assessed by comparing the expression profile of KIF11,KOC1, GHSR1b, NTSR1 or FOXM1 gene. In some embodiments, expressionlevels of KIF11 and KOC1 are determined.

Expression Profile

The invention also provides an NSCLC reference expression profilecomprising a pattern of gene expression levels of two or more of KIF11,KOC1, GHSR1b, NTSR1 and FOXM1 genes. The expression profile serves as acontrol for the diagnosis of NSCLC or predisposition for developing thedisease, monitoring the course of treatment and assessing prognosis of asubject with the disease.

Kits of the Invention

The invention also provides a kit comprising two or more detectionreagents, e.g., a nucleic acid that specifically binds to or identifiesone or more of KIF11, KOC1, GHSR1b, NTSR1 and FOXM1 genes. Such nucleicacids specifically binding to or identifying one or more of KIF11, KOC1,GHSR1b, NTSR1 and FOXM1 genes are exemplified by oligonucleotidesequences that are complementary to a portion of KIF11, KOC1, GHSR1b,NTSR1 or FOXM1 polynucleotides or antibodies which bind to polypeptidesencoded by the KIF11, KOC1, GHSR1b, NTSR1 or FOXM1 gene. The reagentsare packaged together in the form of a kit. The reagents, such as anucleic acid or antibody (either bound to a solid matrix or packagedseparately with reagents for binding them to the matrix), a controlreagent (positive and/or negative) and/or a means of detection of thenucleic acid or antibody are preferably packaged in separate containers.Instructions (e.g., written, tape, VCR, CD-ROM, etc.) for carrying outthe assay may be included in the kit. The assay format of the kit may beNorthern hybridization or sandwich ELISA known in the art.

For example, a detection reagent is immobilized on a solid matrix suchas a porous strip to form at least one detection site. The measurementor detection region of the porous strip may include a plurality ofdetection sites, each detection site containing a detection reagent. Atest strip may also contain sites for negative and/or positive controls.Alternatively, control site(s) is located on a separate strip from thetest strip. Optionally, the different detection sites may containdifferent amounts of immobilized reagents, i.e., a higher amount in thefirst detection site and lesser amounts in subsequent sites. Upon theaddition of a test biological sample, the number of sites displaying adetectable signal provides a quantitative indication of the amount ofKIF11, GHSR1b, NTSR1 or FOXM1 gene, or polypeptides encoded by the genepresent in the sample. The detection sites may be configured in anysuitably detectable shape and are typically in the shape of a bar or dotspanning the width of a teststrip.

Alternatively, the kit contains a nucleic acid substrate arraycomprising two or more of the KIF11, GHSR1b, NTSR1, and FOXM1 genesequences. The expression of 2 or 3 of the genes represented by KIF11,GHSR1b, NTSR1, and FOXM1 genes are identified by virtue of the level ofbinding to an array test strip or chip. The substrate array can be on,e.g., a solid substrate, e.g., a “chip” as described in U.S. Pat. No.5,744,305.

In some embodiments, the kits can be used for predicting an NSCLCprognosis. The kits in these embodiments, can comprise a reagent fordetecting mRNA encoding the amino acid sequence of KIF11 or KOC1, areagent for detecting the proteins or reagents for detecting thebiological activity of the KIF11 or KOC1 protein.

The invention also provides kits for the detection of a compound thatregulates RNA transporting activity. The kits may comprise a cellexpressing a KIF11 polypeptide, or functional equivalent, a KOC1polypeptide, or functional equivalent, and RNA to be transported, andDCTN1.

The kits of the invention may also be used to screen for compounds fortreating or preventing NSCLC. The kits may comprise a KOC1 polypeptide,or functional equivalent, and an RNA that is bound by the KOC1polypeptide or functional equivalent. In the present invention, any RNAtransportable with RNA transporter activity of KOC1-KIF11 complex can beused as the RNA to be transported. Prefer RNA can be selected fromtranscripts of genes shown in table 2, or fragment thereof. An RNA to betransported may also be labeled for detecting RNA transporter activity.Furthermore, in the present invention, KOC1 and KIF11 polypeptide orfunctional equivalent thereof is expressed as fusion protein with signalgenerating protein for observation by microscopy or cell imagingsystems. For example, ECFP, EYFP, and EGFP may be used for signalgenerating protein.

Array and Pluralities

The invention also includes a nucleic acid substrate array comprisingone or more of the KIF11, GHSR1b, NTSR1, and FOXM1 genes. The nucleicacids on the array specifically correspond to one or more polynucleotidesequences represented by KIF11, GHSR1b, NTSR1, and FOXM1 genes. Theexpression level of 2, 3 or 4 of the KIF11, GHSR1b, NTSR1, and FOXM1genes is identified by detecting the binding of nucleic acid to thearray.

The invention also includes an isolated plurality (i.e., a mixture oftwo or more nucleic acids) of nucleic acids. The nucleic acids are in aliquid phase or a solid phase, e.g., immobilized on a solid support suchas a nitrocellulose membrane. The plurality includes one or more of thepolynucleotides represented by KIF11, GHSR1b, NTSR1, and FOXM1 genes.According to a further embodiment of the present invention, theplurality includes 2, 3, or 4 of the polynucleotides represented byKIF11, GHSR1b, NTSR1, and FOXM1 genes.

Chips

The DNA chip is a device that is convenient to compare the expressionlevels of a number of genes at the same time. DNA chip-based expressionprofiling can be carried out, for example, by the method as disclosed in“Microarray Biochip Technology” (Mark Schena, Eaton Publishing, 2000),etc.

A DNA chip comprises immobilized high-density probes to detect a numberof genes. Thus, the expression levels of many genes can be estimated atthe same time by a single-round analysis. Namely, the expression profileof a specimen can be determined with a DNA chip. The DNA chip-basedmethod of the present invention comprises the following steps of:

-   -   (1) synthesizing aRNAs or cDNAs corresponding to the marker        genes;    -   (2) hybridizing the aRNAs or cDNAs with probes for marker genes;        and    -   (3) detecting the aRNA or cDNA hybridizing with the probes and        quantifying the amount of mRNA thereof.

The term “aRNA” refers to RNA transcribed from a template cDNA with RNApolymerase. An aRNA transcription kit for DNA chip-based expressionprofiling is commercially available. With such a kit, aRNA can besynthesized from T7 promoter-attached cDNA as a template using T7 RNApolymerase. On the other hand, by PCR using random primer, cDNA can beamplified using as a template a cDNA synthesized from mRNA.

Alternatively, the DNA chip comprises probes, which have been spottedthereon, to detect the marker genes of the present invention (KIF11,GHSR1b, NTSR1 or FOXM1 gene). There is no limitation on the number ofmarker genes spotted on the DNA chip, and 1, 2, 3 or all of the genes,KIF11, GHSR1b, NTSR1, and FOXM1, may be used. Any other genes as well asthe marker genes can be spotted on the DNA chip. For example, a probefor a gene whose expression level is hardly altered may be spotted onthe DNA chip. Such a gene can be used to normalize assay results whenthe assay results are intended to be compared between multiple chips orbetween different assays.

A probe is designed for each marker gene selected, and spotted on a DNAchip. Such a probe may be, for example, an oligonucleotide comprising5-50 nucleotide residues. A method for synthesizing sucholigonucleotides on a DNA chip is known to those skilled in the art.Longer DNAs can be synthesized by PCR or chemically. A method forspotting long DNA, which is synthesized by PCR or the like, onto a glassslide is also known to those skilled in the art. A DNA chip that isobtained by the method as described above can be used for diagnosingNSCLC according to the present invention.

The prepared DNA chip is contacted with aRNA, followed by the detectionof hybridization between the probe and aRNA. The aRNA can be previouslylabeled with a fluorescent dye. A fluorescent dye such as Cy3 (red) andCy5 (green) can be used to label an aRNA. aRNAs from a subject and acontrol are labeled with different fluorescent dyes, respectively. Thedifference in the expression level between the two can be estimatedbased on a difference in the signal intensity. The signal of fluorescentdye on the DNA chip can be detected by a scanner and analyzed using aspecial program. For example, the Suite from Affymetrix is a softwarepackage for DNA chip analysis.

Identifying Compounds that Inhibit NSCLC-Associated Gene Expression

A compound that inhibits the expression or activity of a targetNSCLC-associated gene (KIF11, GHSR1b, NTSR1 or FOXM1 gene) is identifiedby contacting a test cell expressing the NSCLC-associated gene with atest compound and determining the expression level or activity of theNSCLC-associated gene. A decrease in expression compared to the normalcontrol level indicates that the compound is an inhibitor of theNSCLC-associated gene. Such compounds identified according to the methodare useful for inhibiting NSCLC.

The test cell may be a population of cells and includes any cells aslong as the cell expresses the target NSCLC-associated gene(s). Forexample, the test cell may be an immortalized cell line derived from anNSCLC cell. Alternatively, the test cell may be a cell transfected withany of the KIF11, GHSR1b, NTSR1, and FOXM1 genes, or which has beentransfected with the regulatory sequence (e.g., promoter) of any of thegenes that is operably linked to a reporter gene.

Screening Compounds

Using KIF11, GHSR1b, NTSR1 or FOXM1 gene, proteins encoded by the geneor transcriptional regulatory region of the gene, compounds can bescreened that alter the expression of the gene or biological activity ofa polypeptide encoded by the gene. Such compounds are expected to serveas pharmaceuticals for treating or preventing NSCLC.

Therefore, the present invention provides a method of screening for acompound for treating or preventing NSCLC using the polypeptide of thepresent invention. An embodiment of this screening method comprises thesteps of: (a) contacting a test compound with a polypeptide encoded byKIF11, GHSR1b, NTSR1 or FOXM1 gene; (b) detecting the binding activitybetween the polypeptide of the present invention and the test compound;and (c) selecting the compound that binds to the polypeptide.

As explained in more detail below, KOC1 and KIF11 form a complex thathas RNA transporting activity. Thus, the present invention also providesmethods of identifying polypeptides and other compounds that modulateRNA transport activity. For example, a polypeptide can be tested for RNAtransporting activity by contacting a KIF11 polypeptide (SEQ ID NO: 2)or a functional equivalent thereof with an RNA that can be transportedby KIF11 under conditions suitable for transportation of RNA. The levelof RNA transported can be measured using well known techniques, such asby RNA immunoprecipitation, as described in detail below.

A functional equivalent of a KIF11 polypeptide is a polypeptide that hasa biological activity equivalent to a polypeptide consisting of theamino acid sequence of SEQ ID NO: 2 and, for example, comprising theamino acid sequence of SEQ ID NO: 2 (KIF11), wherein one or more aminoacids (usually less than five) are substituted, deleted, or inserted.Alternatively, the polypeptide may be one that comprises an amino acidsequence having at least about 80% homology (also referred to assequence identity) to SEQ ID NO: 2. In other embodiments, thepolypeptide can be encoded by a polynucleotide that hybridizes understringent conditions (as defined below) to a polynucleotide consistingof the nucleotide sequence of SEQ ID NO: 1.

In some embodiments, the KIF11 polypeptide or functional equivalent iscontacted with a KOC1 polypeptide or functional equivalent thereof. Afunctional equivalent of a KOC1 polypeptide is a polypeptide that has abiological activity equivalent to a polypeptide consisting of the aminoacid sequence of SEQ ID NO: 105 and, for example, comprising the aminoacid sequence of SEQ ID NO: 105, wherein one or more amino acids(usually less than five) are substituted, deleted, or inserted.Alternatively, the polypeptide may be one that comprises an amino acidsequence having at least about 80% homology (also referred to assequence identity) to SEQ ID NO: 105. In other embodiments, thepolypeptide can be encoded by a polynucleotide that hybridizes understringent conditions (as defined below) to a polynucleotide consistingof the nucleotide sequence of SEQ ID NO: 104. In some embodiments, afunctional equivalent comprises at least one RRM or KH domain.

The invention also provides methods of identifying agents that modulateRNA transporting activity. In these methods, an agent suspected ofmodulating RNA transporting activity with a KIF11 polypeptide orfunctional equivalent. The level of transported RNA is detected andcompared to the level in a control in the absence of the agent.

The polypeptide to be used for the screening may be a recombinantpolypeptide or a protein derived from the nature or a partial peptidethereof. The polypeptide to be contacted with a test compound can be,for example, a purified polypeptide, a soluble protein, a form bound toa carrier or a fusion protein fused with other polypeptides.

As a method of screening for proteins that bind to KIF11, GHSR1b, NTSR1or FOXM1 polypeptide, many methods well known by a person skilled in theart can be used. Such a screening can be conducted by, for example,immunoprecipitation method using methods well known in the art. Theproteins of the invention can be recombinantly produced using standardprocedures. For example, a gene encoding any of the KIF11, GHSR1b,NTSR1, and FOXM1 polypeptides is expressed in animal cells by insertingthe gene into an expression vector for foreign genes, such as pSV2neo,pcDNA I, pcDNA3.1, pCAGGS and pCD8. The promoter to be used for theexpression may be any promoter that can be used commonly and include,for example, the SV40 early promoter (Rigby in Williamson (ed.), GeneticEngineering, vol. 3. Academic Press, London, 83-141 (1982)), the EF-αpromoter (Kim et al., Gene 91: 217-23 (1990)), the CAG promoter (Niwa etal., Gene 108: 193-200 (1991)), the RSV LTR promoter (Cullen, Methods inEnzymology 152: 684-704 (1987)) the SRα promoter (Takebe et al., MolCell Biol 8: 466 (1988)), the CMV immediate early promoter (Seed andAruffo, Proc Natl Acad Sci USA 84: 3365-9 (1987)), the SV40 latepromoter (Gheysen and Fiers, J Mol Appl Genet 1: 385-94 (1982)), theAdenovirus late promoter (Kaufman et al., Mol Cell Biol 9: 946 (1989)),the HSV TK promoter and so on. The introduction of the gene into animalcells to express a foreign gene can be performed according to anymethods, for example, the electroporation method (Chu et al., NucleicAcids Res 15: 1311-26 (1987)), the calcium phosphate method (Chen andOkayama, Mol Cell Biol 7: 2745-52 (1987)), the DEAE dextran method(Lopata et al., Nucleic Acids Res 12: 5707-17 (1984); Sussman andMilman, Mol Cell Biol 4: 1642-3 (1985)), the Lipofectin method(Derijard, B Cell 7: 1025-37 (1994); Lamb et al., Nature Genetics 5:22-30 (1993): Rabindran et al., Science 259: 230-4 (1993)), and so on.The NSC polypeptide can also be expressed as a fusion protein comprisinga recognition site (epitope) of a monoclonal antibody by introducing theepitope of the monoclonal antibody, whose specificity has been revealed,to the N- or C-terminus of the polypeptide. A commercially availableepitope-antibody system can be used (Experimental Medicine 13: 85-90(1995)). Vectors which can express a fusion protein with, for example,β-galactosidase, maltose binding protein, glutathione S-transferase,green florescence protein (GFP), and so on, by the use of its multiplecloning sites are commercially available.

A fusion protein prepared by introducing only small epitopes consistingof several to a dozen amino acids so as not to change the property ofthe original polypeptide by the fusion is also reported. Epitopes, suchas polyhistidine (His-tag), influenza aggregate HA, human c-myc, FLAG,Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10 protein(T7-tag), human simple herpes virus glycoprotein (HSV-tag), E-tag (anepitope on monoclonal phage) and such, and monoclonal antibodiesrecognizing them can be used as the epitope-antibody system forscreening proteins binding to the KIF11, GHSR1b, NTSR1 or FOXM1polypeptide (Experimental Medicine 13: 85-90 (1995)).

In immunoprecipitation, an immune complex is formed by adding theseantibodies to cell lysate prepared using an appropriate detergent. Theimmune complex consists of the KIF11, GHSR1b, NTSR1 or FOXM1polypeptide, a polypeptide comprising the binding ability with thepolypeptide, and an antibody. Immunoprecipitation can be also conductedusing antibodies against the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide,in addition to the use of antibodies against the above epitopes, whichantibodies can be prepared according to conventional methods and may bein any form, such as monoclonal or polyclonal antibodies, and includesantiserum obtained by immunizing an animal such as a rabbit with thepolypeptide, all classes of polyclonal and monoclonal antibodies, aswell as recombinant antibodies (e.g., humanized antibodies).

Specifically, antibodies against KIF11, GHSR1b, NTSR1 or FOXM1polypeptide can be prepared using techniques well known in the art. Forexample, KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide used as an antigen toobtain an antibody may be derived from any animal species, butpreferably is derived from a mammal such as a human, mouse, or rat, morepreferably from a human. The polypeptide used as the antigen can berecombinantly produced or isolated from natural sources. According tothe present invention, the polypeptide to be used as an immunizationantigen may be a complete protein or a partial peptide of the KIF11,GHSR1b, NTSR1 or FOXM1 polypeptide. A partial peptide may comprise, forexample, the amino (N)-terminal or carboxy (C)-terminal fragment of theKIF11, GHSR1b, NTSR1 or FOXM1 polypeptide.

Any mammalian animal may be immunized with the antigen, but preferablythe compatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorpha or Primates areused. Animals of Rodentia include, for example, mouse, rat and hamster.Animals of Lagomorpha include, for example, rabbit. Animals of Primatesinclude, for example, a monkey of Catarrhini (old world monkey) such asMacaca fascicularis, rhesus monkey, sacred baboon and chimpanzees.

Methods for immunizing animals with antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is astandard method for immunization of mammals. More specifically, antigensmay be diluted and suspended in an appropriate amount of phosphatebuffered saline (PBS), physiological saline, etc. If desired, theantigen suspension may be mixed with an appropriate amount of a standardadjuvant, such as Freund's complete adjuvant, made into emulsion, andthen administered to mammalian animals. Preferably, it is followed byseveral administrations of the antigen mixed with an appropriatelyamount of Freund's incomplete adjuvant every 4 to 21 days. Anappropriate carrier may also be used for immunization. Afterimmunization as above, the serum is examined by a standard method for anincrease in the amount of desired antibodies.

Polyclonal antibodies against KIF11, GHSR1b, NTSR1 or FOXM1 polypeptidemay be prepared by collecting blood from the immunized mammal examinedfor the increase of desired antibodies in the serum, and by separatingserum from the blood by any conventional method. Polyclonal antibodiesinclude serum containing the polyclonal antibodies, as well as thefraction containing the polyclonal antibodies may be isolated from theserum. Immunoglobulin G or M can be prepared from a fraction whichrecognizes only the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide using, forexample, an affinity column coupled with the polypeptide, and furtherpurifying this fraction using protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from themammal immunized with the antigen and checked for the increased level ofdesired antibodies in the serum as described above, and are subjected tocell fusion. The immune cells used for cell fusion are preferablyobtained from spleen. Other preferred parental cells to be fused withthe above immunocyte include, for example, myeloma cells of mammalians,and more preferably myeloma cells having an acquired property for theselection of fused cells by drugs.

The above immunocyte and myeloma cells can be fused according to knownmethods, for example, the method of Milstein et al., (Galfre andMilstein, Methods Enzymol 73: 3-46 (1981)).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as HAT medium(hypoxanthine, aminopterin, and thymidine containing medium). The cellculture is typically continued in the HAT medium for several days toseveral weeks, the time being sufficient to allow all the other cells,with the exception of the desired hybridoma (non-fused cells), to die.Then, the standard limiting dilution is performed to screen and clone ahybridoma cell producing the desired antibody.

In addition to the above method, in which a non-human animal isimmunized with an antigen for preparing hybridoma, human lymphocytessuch as those infected by EB virus may be immunized with KIF11, GHSR1b,NTSR1 or FOXM1 polypeptide, cells expressing the polypeptide, or theirlysates in vitro. Then, the immunized lymphocytes are fused withhuman-derived myeloma cells that are capable of indefinitely dividing,such as U266, to yield a hybridoma producing a desired human antibodythat is able to bind to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptidecan be obtained (Unexamined Published Japanese Patent Application No.(JP-A) Sho 63-17688).

The obtained hybridomas are subsequently transplanted into the abdominalcavity of a mouse and the ascites are extracted. The obtained monoclonalantibodies can be purified by, for example, ammonium sulfateprecipitation, a protein A or protein G column, DEAE ion exchangechromatography, or an affinity column to which any of the targetproteins of the present invention (KIF11, GHSR1b, NTSR1, and FOXM1polypeptide) is coupled. The antibody can be used not only in thepresent screening method, but also for purification and detection ofKIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, and serve also as candidatesfor agonists and antagonists of the polypeptide. In addition, thisantibody can be applied to the antibody treatment for diseases relatedto the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide including NSCLC asdescribed infra.

Monoclonal antibodies thus obtained can be also recombinantly preparedusing genetic engineering techniques (see, for example, Borrebaeck andLarrick, Therapeutic Monoclonal Antibodies, published in the UnitedKingdom by MacMillan Publishers LTD (1990)). For example, a DNA encodingan antibody may be cloned from an immune cell, such as a hybridoma or animmunized lymphocyte producing the antibody, inserted into anappropriate vector, and introduced into host cells to prepare arecombinant antibody. Such recombinant antibody can also be used for thepresent screening.

Furthermore, an antibody used in the screening and so on may be afragment of an antibody or modified antibody, so long as it binds to oneor more of KIF11, GHSR1b, NTSR1, and FOXM1 polypeptides. For instance,the antibody fragment may be Fab, F(ab′)₂, Fv, or single chain Fv(scFv), in which Fv fragments from H and L chains are ligated by anappropriate linker (Huston et al., Proc Natl Acad Sci USA 85: 5879-83(1988)). More specifically, an antibody fragment may be generated bytreating an antibody with an enzyme, such as papain or pepsin.Alternatively, a gene encoding the antibody fragment may be constructed,inserted into an expression vector, and expressed in an appropriate hostcell (see, for example, Co et al., J Immunol 152: 2968-76 (1994); Betterand Horwitz, Methods Enzymol 178: 476-96 (1989); Pluckthun and Skerra,Methods Enzymol 178: 497-515 (1989); Lamoyi, Methods Enzymol 121: 652-63(1986); Rousseaux et al., Methods Enzymol 121: 663-9 (1986); Bird andWalker, Trends Biotechnol 9: 132-7 (1991)).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). Modified antibodies can be obtainedthrough chemically modification of an antibody. These modificationmethods are conventional in the field.

Alternatively, an antibody may be obtained as a chimeric antibody,between a variable region derived from nonhuman antibody and theconstant region derived from human antibody, or as a humanized antibody,comprising the complementarity determining region (CDR) derived fromnonhuman antibody, the frame work region (FR) derived from humanantibody, and the constant region. Such antibodies can be prepared usingknown technology.

Humanization can be performed by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody (see e.g.,Verhoeyen et al., Science 239:1534-1536 (1988)). Accordingly, suchhumanized antibodies are chimeric antibodies, wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species.

Fully human antibodies comprising human variable regions in addition tohuman framework and constant regions can also be used. Such antibodiescan be produced using various techniques known in the art. For examplein vitro methods involve use of recombinant libraries of human antibodyfragments displayed on bacteriophage (e.g., Hoogenboom & Winter, J. Mol.Biol. 227:381 (1991), Similarly, human antibodies can be made byintroducing of human immunoglobulin loci into transgenic animals, e.g.,mice in which the endogenous immunoglobulin genes have been partially orcompletely inactivated. This approach is described, e.g., in U.S. Pat.Nos. 6,150,584, 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,661,016.

Antibodies obtained as above may be purified to homogeneity. Forexample, the separation and purification of the antibody can beperformed according to separation and purification methods used forgeneral proteins. For example, the antibody may be separated andisolated by the appropriately selected and combined use of columnchromatographies, such as affinity chromatography, filter,ultrafiltration, salting-out, dialysis, SDS polyacrylamide gelelectrophoresis, isoelectric focusing, and others (Antibodies: ALaboratory Manual. Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988)), but are not limited thereto. A protein A column andprotein G column can be used as the affinity column. Exemplary protein Acolumns to be used include, for example, Hyper D, POROS, and SepharoseF.F. (Pharmacia).

Exemplary chromatography, with the exception of affinity includes, forexample, ion-exchange chromatography, hydrophobic chromatography, gelfiltration, reverse-phase chromatography, adsorption chromatography, andthe like (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press (1996)). The chromatographic procedures can becarried out by liquid-phase chromatography, such as HPLC and FPLC.

An immune complex can be precipitated, for example with Protein Asepharose or Protein G sepharose when the antibody is a mouse IgGantibody. If the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide is preparedas a fusion protein with an epitope, such as GST, an immune complex canbe formed in the same manner as in the use of the antibody against theKIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, using a substancespecifically binding to these epitopes, such as glutathione-Sepharose4B.

Immunoprecipitation can be performed by following or according to, forexample, the methods in the literature (Harlow and Lane, Antibodies,511-52, Cold Spring Harbor Laboratory publications, New York (1988)).

SDS-PAGE is commonly used for analysis of immunoprecipitated proteinsand the bound protein can be analyzed by the molecular weight of theprotein using gels with an appropriate concentration. Since the proteinbound to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide is difficult todetect by a common staining method, such as Coomassie staining or silverstaining, the detection sensitivity for the protein can be improved byculturing cells in culture medium containing radioactive isotope,³⁵S-methionine or ³⁵S-cystein, labeling proteins in the cells, anddetecting the proteins. The target protein can be purified directly fromthe SDS-polyacrylamide gel and its sequence can be determined, when themolecular weight of the protein has been revealed.

As a method for screening proteins binding to any of KIF11, GHSR1b,NTSR1, and FOXM1 polypeptides using the polypeptide, for example,West-Western blotting analysis (Skolnik et al., Cell 65: 83-90 (1991))can be used. Specifically, a protein binding to KIF11, GHSR1b, NTSR1 orFOXM1 polypeptide can be obtained by preparing a cDNA library fromcells, tissues, organs (for example, tissues such as lung cells) orcultured cells (particularly those derived from NSCLC cells) expected toexpress a protein binding to the KIF11, GHSR1b, NTSR1 or FOXM1polypeptide using a phage vector (e.g., ZAP), expressing the protein onLB-agarose, fixing the protein expressed on a filter, reacting thepurified and labeled KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide with theabove filter, and detecting the plaques expressing proteins bound to theKIF11, GHSR1b, NTSR1 or FOXM1 polypeptide according to the label. TheKIF11, GHSR1b, NTSR1 or FOXM1 polypeptide may be labeled by utilizingthe binding between biotin and avidin, or by utilizing an antibody thatspecifically binds to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, ora peptide or polypeptide (for example, GST) that is fused to the KIF11,GHSR1b, NTSR1 or FOXM1 polypeptide. Methods using radioisotope orfluorescence and such may be also used.

Alternatively, in another embodiment of the screening method of thepresent invention, a two-hybrid system utilizing cells may be used(“MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid AssayKit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-HybridVector System” (Stratagene); the references “Dalton and Treisman, Cell68: 597-612 (1992)”, “Fields and Sternglanz, Trends Genet 10: 286-92(1994)”).

In the two-hybrid system, KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide isfused to the SRF-binding region or GAL4-binding region and expressed inyeast cells. A cDNA library is prepared from cells expected to express aprotein binding to the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, suchthat the library, when expressed, is fused to the VP16 or GAL4transcriptional activation region. The cDNA library is then introducedinto the above yeast cells and the cDNA derived from the library isisolated from the positive clones detected (when a protein binding tothe polypeptide of the invention is expressed in yeast cells, thebinding of the two activates a reporter gene, making positive clonesdetectable). A protein encoded by the cDNA can be prepared byintroducing the cDNA isolated above to E. coli and expressing theprotein.

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,luciferase gene and such can be used in addition to the HIS3 gene.

A compound binding to KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide can alsobe screened using affinity chromatography. For example, KIF11, GHSR1b,NTSR1 or FOXM1 polypeptide may be immobilized on a carrier of anaffinity column, and a test compound, containing a protein capable ofbinding to KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, is applied to thecolumn. A test compound herein may be, for example, cell extracts, celllysates, etc. After loading the test compound, the column is washed, andcompounds bound to KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide can beprepared.

When the test compound is a protein, the amino acid sequence of theobtained protein is analyzed, an oligo DNA is synthesized based on thesequence, and cDNA libraries are screened using the oligo DNA as a probeto obtain a DNA encoding the protein.

A biosensor using the surface plasmon resonance phenomenon may be usedas a mean for detecting or quantifying the bound compound in the presentinvention. When such a biosensor is used, the interaction between KIF11,GHSR1b, NTSR1 or FOXM1 polypeptide and a test compound can be observedreal-time as a surface plasmon resonance signal, using only a minuteamount of polypeptide and without labeling (for example, BIAcore,Pharmacia). Therefore, it is possible to evaluate the binding betweenKIF11, GHSR1b, NTSR1 or FOXM1 polypeptide and a test compound using abiosensor such as BIAcore.

The methods of screening for molecules that bind when an immobilizedKIF11, GHSR1b, NTSR1 or FOXM1 polypeptide is exposed to syntheticchemical compounds, or natural substance banks or a random phage peptidedisplay library, and the methods of screening using high-throughputbased on combinatorial chemistry techniques (Wrighton et al., Science273: 458-64 (1996); Verdine, Nature 384: 11-13 (1996); Hogan, Nature384: 17-9 (1996)) to isolate not only proteins but chemical compoundsthat bind to KIF11, GHSR1b, NTSR1 or FOXM1 protein (including agonistand antagonist) are well known to one skilled in the art.

Alternatively, the present invention provides a method of screening fora compound for treating or preventing NSCLC using KIF11, GHSR1b, NTSR1or FOXM1 polypeptide comprising the steps as follows:

-   -   (a) contacting a test compound with KIF11, GHSR1b, NTSR1 or        FOXM1 polypeptide;    -   (b) detecting the biological activity of the KIF11, GHSR1b,        NTSR1 or FOXM1 polypeptide of step (a); and    -   (c) selecting a compound that suppresses the biological activity        of the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide in comparison        with the biological activity detected in the absence of the test        compound.

Since proteins encoded by any of the genes of KIF11, GHSR1b, NTSR1, andFOXM1 have the activity of promoting cell proliferation of NSCLC cells,a compound which inhibits this activity of one of these proteins can bescreened using this activity as an index.

Any polypeptides can be used for screening so long as they comprise thebiological activity of KIF11, GHSR1b, NTSR1 or FOXM1 proteins. Suchbiological activity includes cell-proliferating activity and bindingability to other proteins of the proteins encoded by KIF11, GHSR1b,NTSR1 or FOXM1 gene. For example, a human protein encoded by KIF11,GHSR1b, NTSR1 or FOXM1 gene can be used and polypeptides functionallyequivalent to these proteins can also be used. Such polypeptides may beexpressed endogenously or exogenously by cells.

The compound isolated by this screening is a candidate for antagonistsof the KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide. The term “antagonist”refers to molecules that inhibit the function of KIF11, GHSR1b, NTSR1 orFOXM1 polypeptide by binding thereto. Moreover, a compound isolated bythis screening is a candidate for compounds which inhibit the in vivointeraction of KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide with molecules(including DNAs and proteins).

When the biological activity to be detected in the present method iscell proliferation, it can be detected, for example, by preparing cellswhich express KIF11, GHSR1b, NTSR1 or FOXM1 polypeptide, culturing thecells in the presence of a test compound, and determining the speed ofcell proliferation, measuring the cell cycle and such, as well as bymeasuring the colony forming activity.

As discussed in detail above, by controlling the expression levels ofKIF11, GHSR1b, NTSR1 or FOXM1 gene, one can control the onset andprogression of NSCLC. Thus, compounds that may be used in the treatmentor prevention of NSCLC, can be identified through screenings that usethe expression levels of one or more of KIF11, GHSR1b, NTSR1, and FOXM1genes as indices. In the context of the present invention, suchscreening may comprise, for example, the following steps:

-   -   (a) contacting a test compound with a cell expressing one or        more of KIF11, GHSR1b, NTSR1, and FOXM1 genes; and    -   (b) selecting a compound that reduces the expression level of        one or more of the genes in comparison with the expression level        detected in the absence of the test compound.

Cells expressing at least one of KIF11, GHSR1b, NTSR1, and FOXM1 genesinclude, for example, cell lines established from NSCLC cells; suchcells can be used for the above screening of the present invention(e.g., A549, NCI-H226, NCI-H522, LC319). The expression level can beestimated by methods well known to one skilled in the art. In the methodof screening, a compound that reduces the expression level of at leastone of the genes can be selected as candidate agents to be used for thetreatment or prevention of NSCLC.

Alternatively, the screening method of the present invention maycomprise the following steps:

-   -   (a) contacting a test compound with a cell into which a vector        comprising the transcriptional regulatory region of one or more        of the marker genes and a reporter gene that is expressed under        the control of the transcriptional regulatory region has been        introduced, wherein the marker genes are selected from the group        of KIF11, GHSR1b, NTSR1, and FOXM1;    -   (b) measuring the activity of said reporter gene; and    -   (c) selecting a compound that reduces the expression level of        said reporter gene as compared to a control.

Suitable reporter genes and host cells are well known in the art. Thereporter construct required for the screening can be prepared using thetranscriptional regulatory region of a marker gene. When thetranscriptional regulatory region of a marker gene has been known tothose skilled in the art, a reporter construct can be prepared using theprevious sequence information. When the transcriptional regulatoryregion of a marker gene remains unidentified, a nucleotide segmentcontaining the transcriptional regulatory region can be isolated from agenome library based on the nucleotide sequence information of themarker gene (e.g., based the 5′ upstream sequence information).

In a further embodiment of the method of screening for a compound fortreating or preventing NSCLC of the present invention, the methodutilizes the binding ability of KIF11 to KOC1, or GHSR1b or NTSR1 toNMU.

As described above, the present inventors revealed that KOC1 not onlyco-localized with KIF11 in human normal tissues, NSCLCs, and cell lines,but also directly interacted with KIF11 in NSCLC cells in vitro, andthat the treatment of NSCLC cells with siRNAs for KIF11 reduced itsexpression and led to growth suppression. The results suggest thatKOC1-KIF11 signaling affects growth of NSCLC cells. Thus, it is expectedthat the inhibition of the binding between KOC1 and KIF11 leads to thesuppression of cell proliferation, and compounds inhibiting the bindingserve as pharmaceuticals for treating or preventing NSCLCs. Thisscreening method includes the steps of: (a) contacting a KIF11polypeptide or functional equivalent thereof with KOC1, or a functionalequivalent thereof, in the presence of a test compound; (b) detectingthe binding between the polypeptide and KOC1; and (c) selecting the testcompound that inhibits the binding between the polypeptide and KOC1.

Furthermore, as described above, the present inventors revealed GHSR1band NTSR1 as the likely targets for the growth-promoting effect of NMUin lung tumors. The present inventors revealed that NMU-25 bound tothese receptors on the cell surface, and that treatment of NSCLC cellswith siRNAs for GHSR1 or NTSR1 reduced expression of the receptors andled to apoptosis. The results suggest that NMU affects growth of NSCLCcells by acting through GHSR1b and/or NTSR1 (FIG. 14). Thus, it isexpected that the inhibition of binding between GHSR1b or NTSR1 and NMUleads to the suppression of cell proliferation, and compounds inhibitingthe binding serve as pharmaceuticals for treating or preventing NSCLCs.This screening method includes the steps of: (a) contacting a GHSR1b orNTSR1 polypeptide or functional equivalent thereof with NMU in thepresence of a test compound; (b) detecting binding between thepolypeptide and NMU; and (c) selecting the test compound that inhibitsbinding between the polypeptide and NMU.

KOC1 and KIF11 polypeptides, or GHSR1b or NTSR1 and NMU polypeptides tobe used for the screening may be a recombinant polypeptide or a proteinderived from the nature, or may also be a partial peptide thereof solong as it retains the binding ability to each other. Such partialpeptides retaining the binding ability are herein referred to as“functional equivalents”. The KOC1 and KIF11 polypeptides, or GHSR1b orNTSR1 and NMU polypeptides to be used in the screening can be, forexample, a purified polypeptide, a soluble protein, a form bound to acarrier or a fusion protein fused with other polypeptides.

As a method of screening for compounds that inhibit binding between KOC1and KIF11, or GHSR1b or NTSR1 and NMU, many methods well known by oneskilled in the art can be used. Such a screening can be carried out asan in vitro assay system, for example, in a cellular system. Morespecifically, first, either KOC1 or KIF11, or GHSR1b or NTSR1, or NMU isbound to a support, and the other protein is added together with a testcompound thereto. Next, the mixture is incubated, washed and the otherprotein bound to the support is detected and/or measured.

Examples of supports that may be used for binding proteins includeinsoluble polysaccharides, such as agarose, cellulose and dextran; andsynthetic resins, such as polyacrylamide, polystyrene and silicon;preferably commercial available beads and plates (e.g., multi-wellplates, biosensor chip, etc.) prepared from the above materials may beused. When using beads, they may be filled into a column. Alternatively,the use of magnetic beads of also known in the art, and enables toreadily isolate proteins bound on the beads via magnetism.

The binding of a protein to a support may be conducted according toroutine methods, such as chemical bonding and physical adsorption.Alternatively, a protein may be bound to a support via antibodiesspecifically recognizing the protein. Moreover, binding of a protein toa support can be also conducted by means of avidin and biotin.

The binding between proteins is carried out in buffer, for example, butare not limited to, phosphate buffer and Tris buffer, as long as thebuffer does not inhibit binding between the proteins.

In the present invention, a biosensor using the surface plasmonresonance phenomenon may be used as a mean for detecting or quantifyingthe bound protein. When such a biosensor is used, the interactionbetween the proteins can be observed real-time as a surface plasmonresonance signal, using only a minute amount of polypeptide and withoutlabeling (for example, BIAcore, Pharmacia). Therefore, it is possible toevaluate binding between the KOC1 and KIF11, or GHSR1b or NTSR1 and NMUusing a biosensor such as BIAcore.

Alternatively, either KOC1 or KIF11, or GHSR1b or NTSR1, or NMU may belabeled, and the label of the bound protein may be used to detect ormeasure the bound protein. Specifically, after pre-labeling one of theproteins, the labeled protein is contacted with the other protein in thepresence of a test compound, and then bound proteins are detected ormeasured according to the label after washing.

Labeling substances such as radioisotope (e.g., ³H, ¹⁴C, ³²P, ³³P, ³⁵S,¹²⁵I, ¹³¹I), enzymes (e.g., alkaline phosphatase, horseradishperoxidase, β-galactosidase, β-glucosidase), fluorescent substances(e.g., fluorescein isothiosyanete (FITC), rhodamine) and biotin/avidin,may be used for the labeling of a protein in the present method. Whenthe protein is labeled with radioisotope, the detection or measurementcan be carried out by liquid scintillation. Alternatively, proteinslabeled with enzymes can be detected or measured by adding a substrateof the enzyme to detect the enzymatic change of the substrate, such asgeneration of color, with absorptiometer. Further, in case where afluorescent substance is used as the label, the bound protein may bedetected or measured using fluorophotometer.

Furthermore, binding of KOC1 and KIF11, or GHSR1b or NTSR1 and NMU canbe also detected or measured using antibodies to the KOC1 and KIF11, orGHSR1b or NTSR1 and NMU. For example, after contacting the KOC1polypeptide immobilized on a support with a test compound and KIF11, themixture is incubated and washed, and detection or measurement can beconducted using an antibody against KIF11. Alternatively, KIF11 may beimmobilized on a support, and an antibody against KOC1 may be used asthe antibody. When the combination of GHSR1b or NTSR1 and NMU is used,GHSR1b or NTSR1 polypeptide may be immobilized on a support with a testcompound and NMU, the mixture is incubated and washed, and detection ormeasurement can be conducted using an antibody against NMU.Alternatively, NMU may be immobilized on a support, and an antibodyagainst GHSR1b or NTSR1 may be used as the antibody.

In case of using an antibody in the present screening, the antibody ispreferably labeled with one of the labeling substances mentioned above,and detected or measured based on the labeling substance. Alternatively,the antibody against KOC1 or KIF11, or GHSR1b or NTSR1, or NMU may beused as a primary antibody to be detected with a secondary antibody thatis labeled with a labeling substance. Furthermore, the antibody bound tothe protein in the screening of the present invention may be detected ormeasured using protein G or protein A column.

Alternatively, in another embodiment of the screening method of thepresent invention, a two-hybrid system utilizing cells may be used(“MATCHMAKER Two-Hybrid system”, “Mammalian MATCHMAKER Two-Hybrid AssayKit”, “MATCHMAKER one-Hybrid system” (Clontech); “HybriZAP Two-HybridVector System” (Stratagene); the references “Dalton and Treisman, Cell68: 597-612 (1992)”, “Fields and Sternglanz, Trends Genet 10: 286-92(1994)”).

In the two-hybrid system, for example, KOC1 polypeptide is fused to theSRF-binding region or GAL4-binding region and expressed in yeast cells.KIF11 polypeptide that binds to KOC1 polypeptide is fused to the VP16 orGAL4 transcriptional activation region and also expressed in the yeastcells in the existence of a test compound. Alternatively, KIF11polypeptide may be fused to the SRF-binding region or GAL4-bindingregion, and KOC1 polypeptide to the VP16 or GAL4 transcriptionalactivation region. When the combination of GHSR1b or NTSR1 and NMU isused in the two-hybrid system, for example, GHSR1b or NTSR1 polypeptideis fused to the SRF-binding region or GAL4-binding region and expressedin yeast cells. NMU polypeptide that binds to GHSR1b or NTSR1polypeptide is fused to the VP16 or GAL4 transcriptional activationregion and also expressed in the yeast cells in the existence of a testcompound. Alternatively, NMU polypeptide may be fused to the SRF-bindingregion or GAL4-binding region, and GHSR1b or NTSR1 polypeptide to theVP16 or GAL4 transcriptional activation region. When the test compounddoes not inhibit the binding between KOC1 and KIF11, or GHSR1b or NTSR1and NMU, the binding of the two activates a reporter gene, makingpositive clones detectable.

As a reporter gene, for example, Ade2 gene, lacZ gene, CAT gene,luciferase gene and such can be used besides HIS3 gene.

Moreover, when the combination of GHSR1b or NTSR1 and NMU is used in thescreening method, since GHSR1b and NTSR1 are polypeptides naturallyexpressed on the cell surface, in a preferable embodiment of the presentscreening method, the polypeptides are expressed on the surface of aliving cell. When the polypeptides are expressed on the surface of aliving cell, the binding between the polypeptide and NMU can be detectedby methods detecting the autocrine and paracrine signaling leading tostimulation of tumor cell growth (Heasley, Oncogene 20: 1563-1569(2001)). For example, the binding between GHSR1 or NTSR1 polypeptide andNMU can be detected by:

-   (1) detecting the concentration of calcium or cAMP in the cell (e.g.    FLIPR assay, Biochem. Biophys. Res. Commun. 276: 435-438, 2000;    Nature 406: 70-74, 2000; J. Biol. Chem. 275:21068-21074, 2000);-   (2) detecting the activation of the polypeptide;-   (3) detecting the interaction between the polypeptide and G-protein    (e.g. FLIPR assay, Biochem. Biophys. Res. Commun. 276: 435-438,    2000; Nature 406: 70-74, 2000; J. Biol. Chem. 275:21068-21074, 2000,    or binding assay with ¹²⁵I labeled peptide);-   (4) detecting the activation of phospholipase C or its down stream    pathway (Oncogene 20:1563-1569, 2001);-   (5) detecting the activation of kinases of the protein kinase    cascade, such as Raf, MEK, ERKs, and protein kinase D (PKD)    (Oncogene 20:1563-1569, 2001);-   (6) detecting the activation of a member of Src/Tec/Bmx-family of    tyrosine kinases (Oncogene 20:1563-1569, 2001);-   (7) detecting the activation of a member of the Ras and Rho family,    regulation of a member of the JNK members of MAP families, or the    reorganization of the actin cytoskeleton (Oncogene 20:1563-1569,    2001);-   (8) detecting the activation of any signal complex mediated by the    polypeptide activation;-   (9) detecting the change in subcellular localization of the    polypeptide including the ligand-induced internalization/endocytosis    of the polypeptide (J. Cell Sci., 113: 2963-2975, 2000; J.    Histochem. Cytochem. 48:1553-1563, 2000; Endocrinology Oct.    23, 2003. as doi: 10. 1210/en. 2003-0974);-   (10) detecting the activation of any transcription factor downstream    of the polypeptides or the activation of their downstream gene; and-   (11) detecting cell proliferation, transformation, or any other    oncogenic phenotype of the cell.

Any test compound, for example, cell extracts, cell culture supernatant,products of fermenting microorganism, extracts from marine organism,plant extracts, purified or crude proteins, peptides, non-peptidecompounds, synthetic micromolecular compounds and natural compounds canbe used in the screening methods of the present invention. The testcompound of the present invention can be also obtained using any of thenumerous approaches in combinatorial library methods known in the art,including (1) biological libraries, (2) spatially addressable parallelsolid phase or solution phase libraries, (3) synthetic library methodsrequiring deconvolution, (4) the “one-bead one-compound” library methodand (5) synthetic library methods using affinity chromatographyselection. The biological library methods using affinity chromatographyselection is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145(1997)). Examples of methods for the synthesis of molecular librariescan be found in the art (DeWitt et al., Proc. Natl. Acad. Sci. USA 90:6909 (1993); Erb et al., Proc. Natl. Acad. Sci. USA 91: 11422 (1994);Zuckermann et al., J. Med. Chem. 37: 2678 (1994); Cho et al., Science261: 1303 (1993); Carell et al., Angew. Chem. Int. Ed. Engl. 33: 2059(1994); Carell et al., Angew. Chem. Int. Ed. Engl. 33: 2061 (1994);Gallop et al., J. Med. Chem. 37: 1233 (1994)). Libraries of compoundsmay be presented in solution (see Houghten, Bio/Techniques 13: 412(1992)) or on beads (Lam, Nature 354: 82 (1991)), chips (Fodor, Nature364: 555 (1993)), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat.No. 5,571,698;5,403,484, and 5,223,409), plasmids (Cull et al., Proc.Natl. Acad. Sci. USA 89: 1865 (1992)) or phage (Scott and Smith, Science249: 386 (1990); Delvin, Science 249: 404 (1990); Cwirla et al., Proc.Natl. Acad. Sci. USA 87: 6378 (1990); Felici, J. Mol. Biol. 222: 301(1991); US Pat. Application 2002103360). The test compound exposed to acell or protein according to the screening methods of the presentinvention may be a single compound or a combination of compounds. When acombination of compounds are used in the screening methods of theinvention, the compounds may be contacted sequentially orsimultaneously.

A compound isolated by the screening methods of the present invention isa candidate for drugs which inhibit the activity of KIF11, GHSR1b, NTSR1or FOXM1 polypeptide, for treating or preventing diseases attributed to,for example, cell proliferative diseases, such as NSCLC. A compound inwhich a part of the structure of the compound obtained by the presentscreening methods of the present invention is converted by addition,deletion and/or replacement, is included in the compounds obtained bythe screening methods of the present invention. A compound effective insuppressing the expression of over-expressed genes, i.e., KIF11, GHSR1b,NTSR1 or FOXM1 gene, is deemed to have a clinical benefit and can befurther tested for its ability to prevent cancer cell growth in animalmodels or test subjects.

Selecting a Therapeutic Agent for Treating and/or Preventing NSCLC thatis Appropriate for a Particular Individual

Differences in the genetic makeup of individuals can result indifferences in their relative abilities to metabolize various drugs. Acompound that is metabolized in a subject to act as an anti-NSCLC agentcan manifest itself by inducing a change in gene expression pattern inthe subject's cells from that characteristic of a cancerous state to agene expression pattern characteristic of a non-cancerous state.Accordingly, the differentially expressed KIF11, GHSR1b, NTSR1, andFOXM1 genes disclosed herein allow for selection of a putativetherapeutic or prophylactic inhibitor of NSCLC specifically adequate fora subject by testing candidate compounds in a test cell (or test cellpopulation) derived from the selected subject.

To identify an anti-NSCLC agent, that is appropriate for a specificsubject, a test cell or test cell population derived from the subject isexposed to a therapeutic agent and the expression of one or more of theKIF11, GHSR1b, NTSR1, and FOXM1 genes is determined.

The test cell is or the test cell population contains an NSCLC cellexpressing an NSCLC-associated gene. Preferably, the test cell is or thetest cell population contains a lung cell. For example, the test cell ortest cell population is incubated in the presence of a candidate agentand the pattern of gene expression of the test cell or cell populationis measured and compared to one or more reference profiles, e.g., anNSCLC reference expression profile or an non-NSCLC reference expressionprofile.

A decrease in the expression of one or more of KIF11, GHSR1b, NTSR1, andFOXM1 in a test cell or test cell population relative to a referencecell population containing NSCLC is indicative that the agent istherapeutic.

The test agent can be any compound or composition. For example, the testagent is an immunomodulatory agent.

Methods for Treating or Preventing NSCLC

The present invention provides a method for treating, alleviating orpreventing NSCLC in a subject. Therapeutic compounds are administeredprophylactically or therapeutically to subjects suffering from or atrisk of (or susceptible to) developing NSCLC. Such subjects areidentified using standard clinical methods or by detecting an aberrantlevel of expression or activity of KIF11, GHSR1b, NTSR1 or FOXM1 gene orpolypeptide. Prophylactic administration occurs prior to themanifestation of overt clinical symptoms of disease, such that a diseaseor disorder is prevented or alternatively delayed in its progression.

The method includes decreasing the expression or function, or both, ofone or more gene products of genes whose expression is aberrantlyincreased (“over-expressed gene”; KIF11, GHSR1b, NTSR1 or FOXM1 gene) inan NSCLC cell relative to normal cells of the same tissue type fromwhich the NSCLC cells are derived. The expression may be inhibited byany method known in the art. For example, a subject may be treated withan effective amount of a compound that decreases the amount of one ormore of the KIF11, GHSR1b, NTSR1 or FOXM1 gene in the subject.Administration of the compound can be systemic or local. Suchtherapeutic compounds include compounds that decrease the expressionlevel of such gene that endogenously exists in the NSCLC cells (i.e.,compounds that down-regulate the expression of the over-expressedgene(s), KIF11, GHSR1b and/or NTSR1 genes). The administration of suchtherapeutic compounds counter the effects of aberrantly-over expressedgene(s) in the subjects NSCLC cells and are expected to improve theclinical condition of the subject. Such compounds can be obtained by thescreening method of the present invention described above.

The compounds that modulate the activity of a protein encoded by KIF11,GHSR1b, NTSR1 or FOXM1 gene that can be used for treating or preventingNSCLC of the present invention include besides proteins,naturally-occurring cognate ligand of these proteins, peptides,peptidomimetics and other small molecules.

Alternatively, the expression of these over-expressed gene(s) (KIF11,GHSR1b, NTSR1 and/or FOXM1) can be inhibited by administering to thesubject a nucleic acid that inhibits or antagonizes the expression ofthe over-expressed gene(s). Antisense oligonucleotides, siRNAs orribozymes which disrupt the expression of the over-expressed gene(s) canbe used for inhibiting the expression of the over-expressed gene(s).

As noted above, antisense-oligonucleotides corresponding to any of thenucleotide sequence of KIF11, GHSR1b, NTSR1 or FOXM1 gene can be used toreduce the expression level of the gene. Antisense-oligonucleotidescorresponding to KIF11, GHSR1b, NTSR1, and FOXM1 genes that areup-regulated in NSCLC are useful for the treatment or prevention ofNSCLC. Specifically, the antisense-oligonucleotides against the genesmay act by binding to any of the corresponding polypeptides encoded bythese genes, or mRNAs corresponding thereto, thereby inhibiting thetranscription or translation of the genes, promoting the degradation ofthe mRNAs, and/or inhibiting the expression of proteins encoded by theKIF11, GHSR1b, NTSR1, and FOXM1 nucleotides, and finally inhibiting thefunction of the proteins. The term “antisense-oligonucleotides” as usedherein encompasses both nucleotides that are entirely complementary tothe target sequence and those having a mismatch of one or morenucleotides, so long as the antisense-oligonucleotides can specificallyhybridize to the target sequence. For example, theantisense-oligonucleotides of the present invention includepolynucleotides that have a homology (also referred to as sequenceidentity) of at least 70% or higher, preferably at 80% or higher, morepreferably 90% or higher, even more preferably 95% or higher over a spanof at least 15 continuous nucleotides up to the full length sequence ofany of the nucleotide sequences of KIF11, GHSR1b, NTSR1 or FOXM1 gene.Algorithms known in the art can be used to determine the homology.Furthermore, derivatives or modified products of theantisense-oligonucleotides can also be used asantisense-oligonucleotides in the present invention. Examples of suchmodified products include lower alkyl phosphonate modifications such asmethyl-phosphonate-type or ethyl-phosphonate-type, phosphorothioatemodifications and phosphoroamidate modifications.

siRNA molecules of the invention can also be defined by their ability tohybridize specifically to mRNA or cDNA from the genes disclosed here.For the purposes of this invention the terms “hybridize” or “hybridizespecifically” are used to refer the ability of two nucleic acidmolecules to hybridize under “stringent hybridization conditions.” Thephrase “stringent hybridization conditions” refers to conditions underwhich a nucleic acid molecule will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but not detectably toother sequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization. Exemplarystringent hybridization conditions can be as following: 50% formamide,5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubatingat 65° C., with wash in 0.2×SSC, and 0.1% SDS at 50° C. Theantisense-oligonucleotides and derivatives thereof act on cellsproducing the proteins encoded by KIF11, GHSR1b, NTSR1 or FOXM1 gene bybinding to the DNA or mRNA encoding the protein, inhibitingtranscription or translation thereof, promoting the degradation of themRNAs and inhibiting the expression of the protein, thereby resulting inthe inhibition of the protein function.

An antisense-oligonucleotides and derivatives thereof can be made intoan external preparation, such as a liniment or a poultice, by mixingwith a suitable base material which is inactive against the derivative.

The antisense-oligonucleotides of the invention inhibit the expressionof at least one protein encoded by any one of KIF11, GHSR1b, NTSR1, andFOXM1 genes, and thus are useful for suppressing the biological activityof the protein.

The polynucleotides that inhibit one or more gene products ofover-expressed genes also include small interfering RNAs (siRNA)comprising a combination of a sense strand nucleic acid and an antisensestrand nucleic acid of the nucleotide sequence encoding anover-expressed protein encoded by KIF11, GHSR1b, NTSR1 or FOXM1 gene.The term “siRNA” refers to a double stranded RNA molecule which preventstranslation of a target mRNA. Standard techniques of introducing siRNAinto the cell can be used in the treatment or prevention of the presentinvention, including those in which DNA is a template from which RNA istranscribed. The siRNA is constructed such that a single transcript hasboth the sense and complementary antisense sequences from the targetgene, e.g., a hairpin.

The method is used to suppress gene expression of a cell withup-regulated expression of KIF11, GHSR1b, NTSR1 or FOXM1 gene. Bindingof the siRNA to KIF11, GHSR1b, NTSR1 or FOXM1 gene transcript in thetarget cell results in a reduction of KIF11, GHSR1b, NTSR1 or FOXM1protein production by the cell. The length of the oligonucleotide is atleast about 10 nucleotides and may be as long as the naturally occurringtranscript. Preferably, the oligonucleotide is about 19 to about 25nucleotides in length. Most preferably, the oligonucleotide is less thanabout 75, about 50 or about 25 nucleotides in length. Preferable siRNAof the present invention include the polynucleotides having thenucleotide sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37, or 108 as thetarget sequence, which all proved to be effective for suppressing cellviability of NSCLC cell lines. Specifically, a preferable siRNA used inthe present invention has the general formula:

5′-[A]-[B]-[A′]-3′

wherein [A] is a ribonucleotide sequence corresponding to a targetsequence of KIF11, GHSR1b, NTSR1 or FOXM1; [B] is a ribonucleotidesequence consisting of about 3 to about 23 nucleotides; and [A′] is aribonucleotide sequence complementary to [A]. Herein, the phrase a“target sequence of KIF11, GHSR1b, NTSR1 or FOXM1 gene” refers to asequence that, when introduced into NSCLC cell lines, is effective forsuppressing cell viability. Preferred target sequence of KIF11, GHSR1b,NTSR1 or FOXM1 gene includes nucleotide sequences comprising: SEQ IDNOs: 32, 33, 34, 35, 36, 37, and 108. The complementary sequence [A′]and [A] hybridize to each other to form a double strand, and the wholesiRNA molecule with the general formula 5′-[A]-[B]-[A]-3′ forms ahairpin loop structure. As used herein, the term “complementary” refersto a Watson-Crick or Hoogsteen base pairing between nucleotide units ofa polynucleotide, and hybridization or binding of nucleotide unitsindicates physical or chemical interaction between the units underappropriate conditions to form a stable duplex (double-strandedconfiguration) containing few or no mismatches. In a preferredembodiment, such duplexes contain no more than 1 mismatch for every 10base pairs. Particularly preferred duplexes are fully complementary andcontain no mismatch. The siRNA against the mRNA of KIF11, GHSR1b, NTSR1or FOXM1 gene to be used in the present invention contains a targetsequence shorter than the whole mRNA of KIF11, GHSR1b, NTSR1 or FOXM1gene, and has a sequence of 500, 200, or 75 nucleotides as the wholelength. Also included in the invention is a vector containing one ormore of the nucleic acids described herein, and a cell containing thevectors. The isolated nucleic acids of the present invention are usefulfor siRNA against KIF11, GHSR1b, NTSR1 or FOXM1 or DNA encoding thesiRNA. When the nucleic acids are used for siRNA or coding DNA thereof,the sense strand is preferably longer than about 19 nucleotides, andmore preferably longer than about 21 nucleotides.

Furthermore, the nucleotide sequence of siRNAs may be designed using asiRNA design computer program available from the Ambion website (foundon the World Wide Web at ambion.com/techlib/misc/siRNA_finder.html). Thenucleotide sequences for the siRNA are selected by the computer programbased on the following protocol:

Selection of siRNA Target Sites:

-   1. Beginning with the AUG start codon of the transcript, scan    downstream for AA dinucleotide sequences. Record the occurrence of    each AA and the 3′ adjacent 19 nucleotides as potential siRNA target    sites. Tuschl, et al. Genes Dev 13(24): 3191-7 (1999), not recommend    against designing siRNA against the 5′ and 3′ untranslated regions    (UTRs) and regions near the start codon (within 75 bases) as these    may be richer in regulatory protein binding sites, and thus the    complex of endonuclease and siRNAs that were designed against these    regions may interfere with the binding of UTR-binding proteins    and/or translation initiation complexes.-   2. Compare the potential target sites to the human genome database    and eliminate from consideration any target sequences with    significant homology to other coding sequences. The homology search    can be performed using BLAST, which can be found on the NCBI server    at: www.ncbi.nlm.nih.gov/BLAST/3.-   3. Select qualifying target sequences for synthesis. On the website    of Ambion, several preferable target sequences can be selected along    the length of the gene for evaluation.

The siRNAs inhibit the expression of over-expressed KIF11, GHSR1b, NTSR1or FOXM1 protein and is thereby useful for suppressing the biologicalactivity of the protein. Therefore, a composition comprising the siRNAis useful in treating or preventing non-small cell lung cancer.

The nucleic acids that inhibit one or more gene products ofover-expressed genes KIF11, GHSR1b, NTSR1, and FOXM1 also includeribozymes against the gene(s).

The ribozymes inhibit the expression of over-expressed KIF11, GHSR1b,NTSR1 or FOXM1 protein and is thereby useful for suppressing thebiological activity of the protein. Therefore, a composition comprisingthe ribozyme is useful in treating or preventing NSCLC.

Generally, ribozymes are classified into large ribozymes and smallribozymes. A large ribozyme is known as an enzyme that cleaves thephosphate ester bond of nucleic acids. After the reaction with the largeribozyme, the reacted site consists of a 5′-phosphate and 3′-hydroxylgroup. The large ribozyme is further classified into (1) group I intronRNA catalyzing transesterification at the 5′-splice site by guanosine;(2) group II intron RNA catalyzing self-splicing through a two stepreaction via lariat structure; and (3) RNA component of the ribonucleaseP that cleaves the tRNA precursor at the 5′ site through hydrolysis. Onthe other hand, small ribozymes have a smaller size (about 40 bp)compared to the large ribozymes and cleave RNAs to generate a5′-hydroxyl group and a 2′-3′ cyclic phosphate. Hammerhead typeribozymes (Koizumi et al., FEBS Lett. 228: 225 (1988)) and hairpin typeribozymes (Buzayan, Nature 323: 349 (1986); Kikuchi and Sasaki, NucleicAcids Res. 19: 6751 (1992)) are included in the small ribozymes. Methodsfor designing and constructing ribozymes are known in the art (seeKoizumi et al., FEBS Lett. 228: 225 (1988); Koizumi et al., NucleicAcids Res. 17: 7059 (1989); Kikuchi and Sasaki, Nucleic Acids Res. 19:6751 (1992)) and ribozymes inhibiting the expression of anover-expressed NSC protein can be constructed based on the sequenceinformation of the nucleotide sequence encoding KIF11, GHSR1b, NTSR1 orFOXM1 protein according to conventional methods for producing ribozymes.

The ribozymes inhibit the expression of over-expressed KIF11, GHSR1b,NTSR1 or FOXM1 protein and is thereby useful for suppressing thebiological activity of the protein. Therefore, a composition comprisingthe ribozyme is useful in treating or preventing NSCLC.

Alternatively, the function of one or more gene products of theover-expressed genes is inhibited by administering a compound that bindsto or otherwise inhibits the function of the gene products. For example,the compound is an antibody which binds to the over-expressed geneproduct or gene products.

The present invention refers to the use of antibodies, particularlyantibodies against a protein encoded by any of the up-regulated genesKIF11, GHSR1b, NTSR1 or FOXM1, or a fragment of the antibody. As usedherein, the term “antibody” refers to an immunoglobulin molecule havinga specific structure that interacts (binds) specifically with a moleculecomprising the antigen used for synthesizing the antibody (i.e., theup-regulated gene product) or with an antigen closely related to it. Anantibody that binds to the over-expressed KIF11, GHSR1b, NTSR1 or FOXM1nucleotide may be in any form, such as monoclonal or polyclonalantibodies, and includes antiserum obtained by immunizing an animal suchas a rabbit with the polypeptide, all classes of polyclonal andmonoclonal antibodies, human antibodies and humanized antibodiesproduced by genetic recombination. Furthermore, the antibody used in themethod of treating or preventing NSCLC of the present invention may be afragment of an antibody or a modified antibody, so long as it binds toone or more of the proteins encoded by the marker genes (KIF11, GHSR1b,NTSR1 or FOXM1 gene). The antibodies and antibody fragments used in thepresent method of treating or preventing NSCLC may be modified, andinclude chemically modified and chimeric antibodies. Such antibodies andantibody fragments can be obtained according to the above-mentionedmethods, supra.

When the obtained antibody is to be administered to the human body(antibody treatment), a human antibody or a humanized antibody ispreferable for reducing immunogenicity.

For example, transgenic animals having a repertory of human antibodygenes may be immunized with an antigen such as KIF11, GHSR1b, NTSR1 orFOXM1 polypeptide, cells expressing the polypeptide, or their lysates.Antibody producing cells are then collected from the animals and fusedwith myeloma cells to obtain hybridoma, from which human antibodiesagainst the polypeptide can be prepared (see WO92-03918, WO93-2227,WO94-02602, WO94-25585, WO96-33735, and WO96-34096).

Alternatively, an immune cell, such as an immunized lymphocyte,producing antibodies may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

The present invention provides a method for treating or preventingNSCLC, using an antibody against an over-expressed KIF11, GHSR1b, NTSR1or FOXM1 polypeptide. According to the method, a pharmaceuticallyeffective amount of an antibody against KIF11, GHSR1b, NTSR1 or FOXM1polypeptide is administered. An antibody against an over-expressedKIF11, GHSR1b, NTSR1 or FOXM1 polypeptide is administered at a dosagesufficient to reduce the activity of KIF11, GHSR1b, NTSR1 or FOXM1protein. Alternatively, an antibody binding to a cell surface markerspecific for tumor cells can be used as a tool for drug delivery. Thus,for example, an antibody against an over-expressed KIF11, GHSR1b, NTSR1or FOXM1 polypeptide conjugated with a cytotoxic agent may beadministered at a dosage sufficient to injure tumor cells.

In addition, dominant negative mutants of the proteins disclosed herecan be used to treat or prevent NSCLC. For example, fragments of KOC1that specifically bind KIF11 can be used. As used here “dominantnegative fragment of KOC1” is a mutated form of KOC1 that is capable ofcomplexing with either of KIF11 and RNA to be transported such that theRNA transporter activity of the complex is diminished. Thus, a dominantnegative fragment is one that is not functionally equivalent to the fulllength KOC1 polypeptide. Preferred dominant negative fragments are thosethat comprise at least one RRM domain of KOC1. Alternatively, in anotherembodiment, the dominant negative fragments have two RRM domains andzero to three of KH domains. For example KOC1DEL2 (2×RRM+2×KH) andKOC1DEL3 (2×RRM without KH) are preferable fragment for dominantnegative effect. The amino acid sequences of KOC1DEL2 and KOC1DEL3consist of positions 1 to 406 and 1-197 of SEQ ID NO:105, respectively.The fragments are typically less than about 300 amino acids, typicallyless than about 200 amino acids.

The present invention also relates to a method of treating or preventingNSCLC in a subject comprising administering to said subject a vaccinecomprising a polypeptide encoded by a nucleic acid selected from thegroup consisting of KIF11, GHSR1b, NTSR1, and FOXM1 genes or animmunologically active fragment of said polypeptide, or a polynucleotideencoding the polypeptide or the fragment thereof. Administration of thepolypeptide induces an anti-tumor immunity in a subject. Thus, thepresent invention further provides a method for inducing anti tumorimmunity. The polypeptide or the immunologically active fragmentsthereof are useful as vaccines against NSCLC. In some cases the proteinsor fragments thereof may be administered in a form bound to the T cellreceptor (TCR) or presented on an antigen presenting cell (APC), such asmacrophage, dendritic cell (DC) or B-cells. Due to the strong antigenpresenting ability of DC, the use of DC is most preferable among theAPCs.

In the present invention, the phrase “vaccine against NSCLC” refers to asubstance that has the function to induce anti-tumor immunity orimmunity to suppress NSCLC upon inoculation into animals. In general,anti-tumor immunity includes immune responses such as follows:

-   -   induction of cytotoxic lymphocytes against tumors,    -   induction of antibodies that recognize tumors, and    -   induction of anti-tumor cytokine production.

Therefore, when a certain protein induces any one of these immuneresponses upon inoculation into an animal, the protein is decided tohave anti-tumor immunity inducing effect. The induction of theanti-tumor immunity by a protein can be detected by observing in vivo orin vitro the response of the immune system in the host against theprotein.

For example, a method for detecting the induction of cytotoxic Tlymphocytes is well known. A foreign substance that enters the livingbody is presented to T cells and B cells by the action of antigenpresenting cells (APCs). T cells that respond to the antigen presentedby APC in antigen specific manner differentiate into cytotoxic T cells(or cytotoxic T lymphocytes; CTLs) due to stimulation by the antigen,and then proliferate (this is referred to as activation of T cells).Therefore, CTL induction by a certain peptide can be evaluated bypresenting the peptide to T cell by APC, and detecting the induction ofCTL. Furthermore, APC has the effect of activating CD4+ T cells, CD8+ Tcells, macrophages, eosinophils and NK cells. Since CD4+ T cells arealso important in anti-tumor immunity, the anti-tumor immunity inducingaction of the peptide can be evaluated using the activation effect ofthese cells as indicators.

A method for evaluating the inducing action of CTL using dendritic cells(DCs) as APC is well known in the art. DC is a representative APC havingthe strongest CTL inducing action among APCs. In this method, the testpolypeptide is initially contacted with DC and then this DC is contactedwith T cells. Detection of T cells having cytotoxic effects against thecells of interest after the contact with DC shows that the testpolypeptide has an activity of inducing the cytotoxic T cells. Activityof CTL against tumors can be detected, for example, using the lysis of⁵¹Cr-labeled tumor cells as the indicator. Alternatively, the method ofevaluating the degree of tumor cell damage using ³H-thymidine uptakeactivity or LDH (lactose dehydrogenase)-release as the indicator is alsowell known.

Apart from DC, peripheral blood mononuclear cells (PBMCs) may also beused as the APC. The induction of CTL is reported to be enhanced byculturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL isshown to be induced by culturing PBMC in the presence of keyhole limpethemocyanin (KLH) and IL-7.

The test polypeptides confirmed to possess CTL inducing activity bythese methods are polypeptides having DC activation effect andsubsequent CTL inducing activity. Therefore, polypeptides that induceCTL against tumor cells are useful as vaccines against NSCLC.Furthermore, APC that acquired the ability to induce CTL against NSCLCby contacting with the polypeptides are useful as vaccines againstNSCLC. Furthermore, CTL that acquired cytotoxicity due to presentationof the polypeptide antigens by APC can be also used as vaccines againstNSCLC. Such therapeutic methods for NSCLC using anti-tumor immunity dueto APC and CTL are referred to as cellular immunotherapy.

Generally, when using a polypeptide for cellular immunotherapy,efficiency of the CTL-induction is known to increase by combining aplurality of polypeptides having different structures and contactingthem with DC. Therefore, when stimulating DC with protein fragments, itis advantageous to use a mixture of multiple types of fragments.

Alternatively, the induction of anti-tumor immunity by a polypeptide canbe confirmed by observing the induction of antibody production againsttumors. For example, when antibodies against a polypeptide are inducedin a laboratory animal immunized with the polypeptide, and when growth,proliferation or metastasis of tumor cells is suppressed by thoseantibodies, the polypeptide can be determined to have an ability toinduce anti-tumor immunity.

Anti-tumor immunity is induced by administering the vaccine of thisinvention, and the induction of anti-tumor immunity enables treatmentand prevention of NSCLC. Therapy against or prevention of the onset ofNSCLC includes any of the steps, such as inhibition of the growth ofNSCLC cells, involution of NSCLC cells and suppression of occurrence ofNSCLC cells. Decrease in mortality of individuals having NSCLC, decreaseof marker genes (in addition to KIF11, GHSR1b and/or NTSR1 genes) in theblood, alleviation of detectable symptoms accompanying NSCLC and suchare also included in the therapy or prevention of NSCLC. Suchtherapeutic and preventive effects are preferably statisticallysignificant. For example, in observation, at a significance level of 5%or less, wherein the therapeutic or preventive effect of a vaccineagainst NSCLC is compared to a control without vaccine administration.For example, Student's t-test, the Mann-Whitney U-test or ANOVA may beused for statistical analysis.

The above-mentioned protein having immunological activity, or apolynucleotide or vector encoding the protein may be combined with anadjuvant. An adjuvant refers to a compound that enhances the immuneresponse against the protein when administered together (orsuccessively) with the protein having immunological activity. Examplesof adjuvants include cholera toxin, salmonella toxin, alum and such, butare not limited thereto. Furthermore, the vaccine of this invention maybe combined appropriately with a pharmaceutically acceptable carrier.Examples of such carriers are sterilized water, physiological saline,phosphate buffer, culture fluid and such. Furthermore, the vaccine maycontain as necessary, stabilizers, suspensions, preservatives,surfactants and such. The vaccine is administered systemically orlocally. Vaccine administration may be performed by singleadministration or boosted by multiple administrations.

When using APC or CTL as the vaccine of this invention, NSCLC can betreated or prevented, for example, by the ex vivo method. Morespecifically, PBMCs of the subject receiving treatment or prevention arecollected, the cells are contacted with the polypeptide ex vivo, andfollowing the induction of APC or CTL, the cells may be administered tothe subject. APC can be also induced by introducing a vector encodingthe polypeptide into PBMCs ex vivo. APC or CTL induced in vitro can becloned prior to administration. By cloning and growing cells having highactivity of damaging target cells, cellular immunotherapy can beperformed more effectively. Furthermore, APC and CTL isolated in thismanner may be used for cellular immunotherapy not only againstindividuals from whom the cells are derived, but also against similartypes of diseases in other individuals.

Moreover, the present invention provides a method for treating orpreventing NSCLC in a subject, wherein a compound obtained according toany of the above-described screening methods is administered to thesubject. Any compound that are obtained according to any of thescreening methods of the present invention can be administered to thesubject so long as it decreases the expression or function, or both, ofone or more gene products of KIF11, GHSR1b, NTSR1, and FOXM1 genes.

siRNA and Vectors Encoding Them

Transfection of vectors expressing siRNA for KIF11, GHSR1b, NTSR1 orFOXM1 leads to growth inhibition of NSCLC cells. Thus, it is anotheraspect of the present invention to provide a double-stranded moleculecomprising a sense-strand and antisense-strand which molecule functionsas an siRNA for KIF11, GHSR1b, NTSR1 or FOXM1, and a vector encoding thedouble-stranded molecule.

The double-stranded molecule of the present invention comprises a sensestrand and an antisense strand, wherein the sense strand comprises aribonucleotide sequence corresponding to a KIF11, GHSR1b, NTSR1 or FOXM1target sequence, and wherein the antisense strand comprises aribonucleotide sequence which is complementary to said sense strand,wherein said sense strand and said antisense strand hybridize to eachother to form said double-stranded molecule, and wherein saiddouble-stranded molecule, when introduced into a cell expressing aKIF11, GHSR1b, NTSR1 or FOXM1 gene, inhibits expression of said gene.

The double-stranded molecule of the present invention may be apolynucleotide derived from its original environment (i.e., when it is anaturally occurring molecule, the natural environment), physically orchemically altered from its natural state, or chemically synthesized.According to the present invention, such double-stranded moleculesinclude those composed of DNA, RNA, and derivatives thereof. A DNA issuitably composed of bases such as A, T, C and G, and T is replaced by Uin an RNA.

As described above, the term “complementary” refers to a Watson-Crick orHoogsteen base pairing between nucleotide units of a polynucleotide, andhybridization or binding of nucleotide units indicates physical orchemical interaction between the units under appropriate conditions toform a stable duplex (double-stranded configuration) containing few orno mismatches. In a preferred embodiment, such duplexes contain no morethan 1 mismatch for every 10 base pairs. Particularly preferred duplexesare fully complementary and contain no mismatch.

The double-stranded molecule of the present invention contains aribonucleotide sequence corresponding to a KIF11, GHSR1b, NTSR1 or FOXM1target sequence shorter than the whole mRNA of KIF11, GHSR1b, NTSR1 orFOXM1 gene. Herein, the phrase a “target sequence of KIF11, GHSR1b,NTSR1 or FOXM1 gene” refers to a sequence that, when introduced intoNSCLC cell lines, is effective for suppressing cell viability.Specifically, the target sequence comprises at least about 10, orsuitably about 19 to about 25 contiguous nucleotides from the nucleotidesequences selected from the group of SEQ ID NOs: 1, 3, 5, and 106. Thatis, the sense strand of the present double-stranded molecule consists ofat least about 10 nucleotides, suitably is longer than 19 nucleotides,and more preferably longer than 21 nucleotides. Preferred targetsequences include the sequences of SEQ ID NOs: 32, 33, 34, 35, 36, 37,and 108. The present double-stranded molecule including the sense strandand the antisense strand is an oligonucleotide shorter than about 100,preferably 75, more preferably 50 and most preferably 25 nucleotides inlength. A suitable double-stranded molecule of the present invention isan oligonucleotide of a length of about 19 to about 25 nucleotides.Furthermore, in order to enhance the inhibition activity of the siRNA,nucleotide “u” can be added to 3′ end of the antisense strand of thetarget sequence. The number of “u”s to be added is at least 2, generally2 to 10, preferably 2 to 5. The added “u”s form single strand at the 3′end of the antisense strand of the siRNA. In these embodiments, thesiRNA molecules of the invention are typically modified as describedabove for antisense molecules. Other modifications are also possible,for example, cholesterol-conjugated siRNAs have shown improvedpharmacological properties (Song et al. Nature Med. 9:347-351 (2003):).

Furthermore, the double-stranded molecule of the present invention maybe a single ribonucleotide transcript comprising the sense strand andthe antisense strand linked via a single-stranded ribonucleotidesequence. Namely, the present double-stranded molecule may have thegeneral formula:

5′-[A]-[B]-[A′]-3′

wherein [A] is a ribonucleotide sequence corresponding to a targetsequence of KIF11, GHSR1b, NTSR1 or FOXM1;[B] is a ribonucleotide sequence (loop sequence) consisting of 3 to 23nucleotides; and[A] is a ribonucleotide sequence complementary to [A]. The complementarysequence [A] and [A] hybridize to each other to form a double strand,and the whole siRNA molecule with the general formula 5′-[A]-[B]-[A′]-3′forms a hairpin loop structure.

The region [A] hybridizes to [A], and then a loop consisting of region[B] is formed. The loop sequence can be selected from those described onthe World Wide Web at ambion.com/techlib/tb/tb_(—)506.html, or thosedescribed in Jacque, J.-M. et al., Nature 418: 435-438 (2002).Additional examples of the loop sequence that can be included in thepresent double-stranded molecules include:

CCC, CCACC or CCACACC: Jacque, J. M. et al., Nature, Vol. 418: 435-438(2002);

UUCG: Lee, N. S. et al., Nature Biotechnology 20:500-505 (2002);Fruscoloni, P. et. al., Proc. Natl. Acad. Sci. USA 100(4): 1639-1644(2003); and

UUCAAGAGA: Dykxhoorn, D. M. et al., Nature Reviews Molecular CellBiology 4: 457-467 (2002).

Preferable siRNAs having hairpin loop structure of the present inventionare shown below. In the following structure, the loop sequence can beselected from the group consisting of: CCC, UUCG, CCACC, CCACACC, andUUCAAGAGA. Among these sequences, the most preferable loop sequence isUUCAAGAGA (corresponding to “ttcaagaga” in a DNA):

guuaguguac gaacuggag-[B]-cuccaguuc guacacuaac(for the target sequence of SEQ ID NO: 32);gugucucugu uggagaucu-[B]-agaucucca acagagacac(for the target sequence of SEQ ID NO: 33);gaaggcaguu gaccaacac-[B]-guguugguc aacugccuuc(for the target sequence of SEQ ID NO: 34);ccucuaccug uccagcaug-[B]-caugcugga cagguagagg(for the target sequence of SEQ ID NO: 35);guucaucagc gccaucugg-[B]-ccagauggc gcugaugaac(for the target sequence of SEQ ID NO: 36);ggucgucaua caggucaac-[B]-guugaccug uaugacgacc(for the target sequence of SEQ ID NO: 37); andgcagcagaaa cgaccgaau-[B]-auucggucg uuucugcugc(for the target sequence of SEQ ID NO: 108).

The present invention further provides a vector encoding thedouble-stranded molecule of the present invention. The vector encodes atranscript having a secondary structure and which comprises the sensestrand and the antisense strand, and suitably comprises asingle-stranded ribonucleotide sequence linking said sense strand andsaid antisense strand. The vector preferably comprises a regulatorysequence adjacent to the region encoding the present double-strandedmolecule that directs the expression of the molecule in an adequatecell. For example, the double-stranded molecules of the presentinvention are intracellularly transcribed by cloning their codingsequence into a vector containing, e.g., a RNA pol III transcriptionunit from the small nuclear RNA (snRNA) U6 or the human H1 RNA promoter.

Alternatively, the present vectors are produced, for example, by cloningthe target sequence into an expression vector so the objective sequenceis operatively-linked to a regulatory sequence of the vector in a mannerto allow expression thereof (transcription of the DNA molecule) (Lee, N.S. et al., Nature Biotechnology 20: 500-505 (2002)). For example, thetranscription of an RNA molecule having an antisense sequence to thetarget sequence is driven by a first promoter (e.g., a promoter sequencelinked to the 3′-end of the cloned DNA) and that having the sense strandto the target sequence by a second promoter (e.g., a promoter sequencelinked to the 5′-end of the cloned DNA). The expressed sense andantisense strands hybridize to each other in vivo to generate a siRNAconstruct to silence a gene that comprises the target sequence.Furthermore, two constructs (vectors) may be utilized to respectivelyproduce the sense and anti-sense strands of a siRNA construct.

For introducing the vectors into a cell, transfection-enhancing agentcan be used. FuGENE (Rochediagnostices), Lipofectamine 2000(Invitrogen), Oligofectamine (Invitrogen), and Nucleofector (Wako pureChemical) are useful as the transfection-enhancing agent.

Pharmaceutical Compositions for Treating or Preventing NSCLC

The present invention provides compositions for treating or preventingNSCLC comprising a compound selected by the present method of screeningfor a compound that alters the expression or activity of anNSCLC-associated gene.

When administering a compound isolated by the screening method of thepresent invention as a pharmaceutical for humans and other mammals, suchas mice, rats, guinea-pig, rabbits, cats, dogs, sheep, pigs, cattle,monkeys, baboons or chimpanzees for treating a cell proliferativedisease (e.g., non-small cell lung cancer), the isolated compound can bedirectly administered or can be formulated into a dosage form usingconventional pharmaceutical preparation methods. Such pharmaceuticalformulations of the present compositions include those suitable fororal, rectal, nasal, topical (including buccal and sub-lingual), vaginalor parenteral (including intramuscular, sub-cutaneous and intravenous)administration, or for administration by inhalation or insufflation. Theformulations are optionally packaged in discrete dosage units.

Pharmaceutical formulations suitable for oral administration includecapsules, cachets or tablets, each containing a predetermined amount ofthe active ingredient. Formulations also include powders, granules,solutions, suspensions or emulsions. The active ingredient is optionallyadministered as a bolus electuary or paste. Tablets and capsules fororal administration may contain conventional excipients such as bindingagents, fillers, lubricants, disintegrant or wetting agents. A tabletmay be made by compression or molding, optionally with one or moreformulational ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredients in afree-flowing form such as powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Molded tablets may be made via molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may be coated according to methods wellknown in the art. Oral fluid preparations may be in the form of, forexample, aqueous or oily suspensions, solutions, emulsions, syrups orelixirs, or may be presented as a dry product for reconstitution withwater or other suitable vehicle prior to use. Such liquid preparationsmay contain conventional additives such as suspending agents,emulsifying agents, non-aqueous vehicles (which may include edible oils)or preservatives. The tablets may optionally be formulated so as toprovide slow or controlled release of the active ingredient in vivo. Apackage of tablets may contain one tablet to be taken on each of themonth. The formulation or dose of medicament in these preparations makesa suitable dosage within the indicated range acquirable.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, saline, water-for-injection,immediately prior to use. Alternatively, the formulations may bepresented for continuous infusion. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtablets of the kind previously described.

Formulations for rectal administration include suppositories withstandard carriers such as cocoa butter or polyethylene glycol.Formulations for topical administration in the mouth, for example,buccally or sublingually, include lozenges, which contain the activeingredient in a flavored base such as sucrose and acacia or tragacanth,and pastilles comprising the active ingredient in a base such asgelatin, glycerin, sucrose or acacia. For intra-nasal administration ofan active ingredient, a liquid spray or dispersible powder or in theform of drops may be used. Drops may be formulated with an aqueous ornon-aqueous base also comprising one or more dispersing agents,solubilizing agents or suspending agents.

For administration by inhalation the compositions are convenientlydelivered from an insufflator, nebulizer, pressurized packs or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecompositions may take the form of a dry powder composition, for example,a powder mix of an active ingredient and a suitable powder base such aslactose or starch. The powder composition may be presented in unitdosage form in, for example, capsules, cartridges, gelatin or blisterpacks from which the powder may be administered with the aid of aninhalator or insufflators.

Other formulations include implantable devices and adhesive patches;which release a therapeutic agent.

When desired, the above-described formulations, adapted to givesustained release of the active ingredient, may be employed. Thepharmaceutical compositions may also contain other active ingredientssuch as antimicrobial agents, immunosuppressants or preservatives.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude flavoring agents.

Preferred unit dosage formulations are those containing an effectivedose, as recited below, of the active ingredient or an appropriatefraction thereof.

For each of the aforementioned conditions, the compositions, e.g.,polypeptides and organic compounds are administered orally or viainjection at a dose of from about 0.1 to about 250 mg/kg per day. Thedose range for adult humans is generally from about 5 mg to about 17.5g/day, preferably about 5 mg to about 10 g/day, and most preferablyabout 100 mg to about 3 g/day. Tablets or other unit dosage forms ofpresentation provided in discrete units may conveniently contain anamount which is effective at such dosage or as a multiple of the same,for instance, units containing about 5 mg to about 500 mg, usually fromabout 100 mg to about 500 mg.

The dose employed will depend upon a number of factors, including theage and sex of the subject, the precise disorder being treated, and itsseverity. Also the route of administration may vary depending upon thecondition and its severity.

The present invention further provides a composition for treating orpreventing NSCLC comprising active ingredient that inhibits theexpression of any one of the gene selected from the group of KIF11,GHSR1b, NTSR1, and FOXM1 genes. Such active ingredient can be anantisense-oligonucleotide, siRNA or ribozyme against the gene, orderivatives, such as expression vector, of theantisense-oligonucleotide, siRNA or ribozyme. The active ingredient maybe made into an external preparation, such as liniment or a poultice, bymixing with a suitable base material which is inactive against thederivatives.

Also, as needed, the active ingredient can be formulated into tablets,powders, granules, capsules, liposome capsules, injections, solutions,nose-drops and freeze-drying agents by adding excipients, isotonicagents, solubilizers, preservatives, pain-killers and such. These can beprepared according to conventional methods for preparing nucleic acidcontaining pharmaceuticals.

Preferably, the antisense-oligonucleotide derivative, siRNA derivativeor ribozyme derivative is given to the patient by direct application tothe ailing site or by injection into a blood vessel so that it willreach the site of ailment. A mounting medium can also be used in thecomposition to increase durability and membrane-permiability. Examplesof mounting mediums include liposome, poly-L-lysine, lipid, cholesterol,lipofectin and derivatives thereof.

The dosage of such compositions can be adjusted suitably according tothe patient's condition and used in desired amounts. For example, a doserange of 0.1 to 100 mg/kg, preferably 0.1 to 50 mg/kg can beadministered.

Another embodiment of the present invention is a composition fortreating or preventing NSCLC comprising an antibody against apolypeptide encoded by any one of the genes selected from the group ofKIF11, GHSR1b, NTSR1, and FOXM1 genes or fragments of the antibody thatbind to the polypeptide.

Although there are some differences according to the symptoms, the doseof an antibody or fragments thereof for treating or preventing NSCLC isabout 0.1 mg to about 100 mg per day, preferably about 1.0 mg to about50 mg per day and more preferably about 1.0 mg to about 20 mg per day,when administered orally to a normal adult (weight 60 kg).

When administering parenterally, in the form of an injection to a normaladult (weight 60 kg), although there are some differences according tothe condition of the patient, symptoms of the disease and method ofadministration, it is convenient to intravenously inject a dose of about0.01 mg to about 30 mg per day, preferably about 0.1 to about 20 mg perday and more preferably about 0.1 to about 10 mg per day. Also, in thecase of other animals too, it is possible to administer an amountconverted to 60 kg of body-weight.

The following examples are presented to illustrate the present inventionand to assist one of ordinary skill in making and using the same. Theexamples are not intended in any way to otherwise limit the scope of theinvention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. Any patents, patent applications andpublications sited herein are incorporated by reference.

BEST MODE FOR CARRYING OUT THE INVENTION Materials and Methods (1)Patients and Tissue Samples

Primary NSCLC samples, of which 22 were classified as adenocarcinomas(ADCs), 14 as squamous-cell carcinomas (SCCs), and one as adenosquamouscarcinoma, had been obtained earlier with informed consent from 37patients (Kikuchi, T. et al., Oncogene 22, 2192-2205 (2003)). Fifteenadditional primary NSCLCs, including seven ADCs and eight SCCs, wereobtained along with adjacent normal lung tissue samples from patientsundergoing surgery at our institutes.

(2) Cell Lines

The 30 human NSCLC and four SCLC cell lines used in this study were asfollows: adenocarcinomas (ADCs) A427, A549, NCI-H23, NCI-H522, LC174,LC176, LC319, PC3, PC9, PC14, PC14-PE6, NCI-H1373, NCI-H1435, NCI-H1793,SK-LU-1, NCI-H358, NCI-H1650 and SW1573; adenosquamous carcinomas (ASCs)NCI-H226, NCI-H596 and NCI-H647; squamous-cell carcinomas (SCCs)RERF-LC-AI, SW-900, SK-MES-1, EBC-1, LU61, NCI-H520, NCI-H1703, andNCI-H2170; large-cell carcinoma (LCC) LX1; and SCLCs DMS114, DMS273,SBC-3, and SBC-5. Human small airway epithelial cells, SAEC were grownin optimized medium (SAGM) purchased from Cambrex Bio Science Inc. Ahuman bronchial epithelial cell line, BEAS2B cells were also served.

Thirty-four human NSCLC or SCLC cancer cell lines and two normalbronchial epithelium cell lines were grown in monolayers in appropriatemedium supplemented with 5 or 10% fetal bovine serum (see Table 1).

TABLE 1 Cell line name Medium Provider adenocarcinoma (ADC) A427EMEM(10% FBS) ATCC(HTB-53) A549 RPMI-1640(10% FBS) ATCC(CCL-185) NCI-H23RPMI-1640(10% FBS) ATCC(CRL-5800) NCI-H522 RPMI-1640(10% FBS)ATCC(CRL-5810) LC174 RPMI-1640(10% FBS) Aichi Cancer Center LC176RPMI-1640(10% FBS) Aichi Cancer Center LC319 RPMI-1640(10% FBS) AichiCancer Center PC-3 DMEM(10% FBS) Tokushima Univeraity PC-9 DMEM(10% FBS)Tokushima Univeraity PC14 RPMI-1640(10% FBS) Tokushima UniveraityPC14-PE6 RPMI-1640(10% FBS) Tokushima Univeraity NCI-H1373 RPMI-1640(10%FBS) ATCC(CRL-5866) NCI-H1435 F12 + DMEM(5% FBS) + EGF(+) SNU BankNCI-H1793 F12 + DMEM(5% FBS) + Glu SNU Bank SK-LU-1 DMEM(10% FBS) SNUBank BAC NCI-H358 RPMI-1640(10% FBS) SNU Bank BAC NCI-H1650RPMI-1640(10% FBS) ATCC(CRL-5883) BAC SW1573 Leibovitz's L-15(10% FBS)ATCC(CRL-2170) adenosquamous carcinoma (ASCs) NCI-H226 RPMI-1640(10%FBS) ATCC(CRL-5826) NCI-H647 RPMI-1640(10% FBS) ATCC(CRL-5834) NCI-H596RPMI-1640(10% FBS) SNU Bank squamous cell carcinoma (SCC) RERF-LC-AlDMEM(10% FBS) Tokushima Univeraity SW-900 Leibovitz's L-15(10% FBS) SNUBank SK-MES-1 DMEM(10% FBS) SNU Bank EBC-1 DMEM(10% FBS) TokushimaUniveraity LU61 DMEM(10% FBS) Central Institute for Experimental AnimalsNCI-H520 RPMI-1640(10% FBS) ATCC(HTB-182) NCI-H1703 RPMI-1640(10% FBS)ATCC(CRL-5889) NCI-H2170 RPMI-1640(10% FBS) ATCC8(CRL-5928) large-cellcarcinoma (LCC) LX1 DMEM(10% FBS) Central Institute for ExperimentalAnimals small-cell lung carcinoma (SCLCs) DMS114 RPMI-1640(10% FBS)ATCC(CRL-2066) DMS273 RPMI-1640(10% FBS) Japanese foundation for cancerresearch SBC-3 RPMI-1640(10% FBS) Tokushima Univeraity SBC-5 EMEM(10%FBS) Tokushima Univeraity small airway epithelial cells SAEC SAGMCambrex Bio Science Inc. human bronchial cell line BEAS2B RPMI-1640(10%FBS) ATCC(CRL-9609)

(3) Semiquantitative RT-PCR Analysis

Total RNA was extracted from cultured cells and clinical tissues usingTrizol reagent (Life Technologies, Inc.) according to the manufacturer'sprotocol. Extracted RNAs and normal human tissue poly(A) RNAs weretreated with DNase I (Nippon Gene) and reverse-transcribed usingoligo(dT) primer and SuperScript II reverse transcriptase (Invitrogen).Semiquantitative RT-PCR experiments were carried out with the followingsynthesized gene-specific primers or with beta-actin (ACTB)-specificprimers as an internal control:

(SEQ. ID. NO. 7) KOC1, 5′-TAAATGGCTTCAGGAGACTTCAG-3′ and(SEQ. ID. NO. 8) 5′-GGTTTTAAATGCAGCTCCTATGTG-3′; (SEQ. ID. NO. 9) KIF11,5′-CTGAACAGTGGGTATCTTCCTTA-3′ and (SEQ. ID. NO. 10)5′-GATGGCTCTTGACTTAGAGGTTC-3′; (SEQ. ID. NO. 11) NMU,5′-TGAAGAGATTCAGAGTGGACGA-3′ and (SEQ. ID. NO. 12)5′-ACTGAGAACATTGACAACACAGG-3′; (SEQ. ID. NO. 13) NMU1R,5′-AAGAGGGACAGGGACAAGTAGT-3′ and (SEQ. ID. NO. 14)5′-ATGCCACTGTTACTGCTTCAG-3′; (SEQ. ID. NO. 15) NMU2R,5′-GGCTCTTACAACTCATGTACCCA-3′ and (SEQ. ID. NO. 16)5′-TGATACAGAGACATGAAGTGAGCA-3′; (SEQ. ID. NO. 17) GHSR1a,5′-TGGTGTTTGCCTTCATCCT-3′ and (SEQ. ID. NO. 18)5′-GAATCCCAGAAGTCTGAACA-3′; (SEQ. ID. NO. 19) GHSR1b,5′-ACGGTCCTCTACAGTCTCA-3′ and (SEQ. ID. NO. 20)5′-CACAGGGAGAGGATAGGA-3′; (SEQ. ID. NO. 21) NTSR1,5′-AGTGGGCTCAGAGTCTAGCAAAT-3′ and (SEQ. ID. NO. 22)5′-TATTGAGAGATACACGGGGTTTG-3′; (SEQ. ID. NO. 23) GHRL,5′-TGAGCCCTGAACACCAGAGAG-3′ and (SEQ. ID. NO. 24)5′-AAAGCCAGATGAGCGCTTCTA-3′; (SEQ. ID. NO. 25) NTS,5′-TCTTCAGCATGATGTGTTGTGT-3′ and (SEQ. ID. NO. 26)5′-TGAGAGATTCATGAGGAAGTCTTG-3′; (SEQ. ID. NO. 27) ACTB,5′-GAGGTGATAGCATTGCTTTCG-3′ and (SEQ. ID. NO. 28)5′-CAAGTCAGTGTACAGGTAAGC-3′.

PCR reactions were optimized for the number of cycles to ensure productintensity within the logarithmic phase of amplification.

Quantitative Real-Time RT-PCR (QRT-PCR) Analysis and Northern-BlotAnalyses

Expression levels of the KOC1 and KIF11 genes were measured by QRT-PCRusing the ABI Prism 7700 sequence detection system (Applied Biosystems).Total RNA was extracted from cultured cells and clinical tissues usingTrizol reagent (Life Technologies, Inc.) according to the manufacturer'sprotocol. Extracted RNAs and normal human tissue poly(A) RNAs weretreated with DNase I (Nippon Gene) and were reverse-transcribed usingoligo (dT) primer and SuperScript II reverse transcriptase (Invitrogen).The TaqMan Pre-Developed Assay Human ACTB (Applied Biosystems;#4333762F) was used for ACTB gene as a quantitative control. A primerpair and a TaqMan probe for each gene were designed by using PrimerExpress software as follows:

(SEQ. ID. NO. 98) KOC1, 5′-ACGAACTCATTTGCTCACTCCTT-3′ (sense),(SEQ. ID. NO. 99) 5′-ACCCACACCCAACACAATTGT-3′ (antisense),(SEQ. ID. NO. 100) 5′-ACAGCAAAGCCC-3′ (TaqMan-MGB probe);(SEQ. ID. NO. 101) KIF11, 5′-TTCACCCTGACAGAGTTCACAAA-3′ (sense)(SEQ. ID. NO. 102) 5′-GGGTGGTCTCCCATAATAGCAA-3′ (antisense),(SEQ. ID. NO. 103) 5′-AGCCCACTTTAGAGTATAC-3′ (TaqMan-MGB probe).

PCR for each gene and the ACTB gene was performed in a single tube induplicate. Results were expressed as the average of these twoindependent tests.

(4) Northern-Blot Analysis

Human multiple-tissue blots (BD Biosciences Clontech) were hybridizedwith ³²P-labeled PCR products of KOC1, KIF11 and GHSR1. cDNA probes ofKOC1, KIF11 and GHSR1 were prepared by RT-PCR using primers similarly asabove. Prehybridization, hybridization, and washing were performedaccording to the supplier's recommendations. The blots wereautoradiographed with intensifying BAS screens (BIO-RAD) at roomtemperature (RT) for 30 to 168 hours.

Generation of Anti-KOC1 and -KIF11 Antibodies

Plasmids expressing KOC1 (full-length) and KIF11 (partial amino acidsequence corresponding to codons 361-1056), each containing His-taggedepitope at the N-terminal, were prepared using pET28 vector (Novagen).Recombinant proteins were expressed in Escherichia coli BL21 codon-plusstrain (Stratagene), purified using TALON resin (BD BiosciencesClontech) according to the supplier's protocol, and inoculated intorabbits. The immune sera were purified on affinity columns according tostandard methodology. Affinity-purified anti-KOC1 and anti-KIF11antibodies were used for western-blot analysis, immunoprecipitation, andimmunostaining We confirmed by western-blot analysis that anti-KOC1antibody are specific to KOC1 and do not cross-react with otherhomologous proteins, IMP-1 and IMP-2 using lysates of NCI-H520 cells,which expressed neither of endogenous IMP-1, -2, and -3, but had beentransfected with HA-tagged IMP-1, -2, and -3 expression vector.

Construction of KOC1 Deletion Mutants and Immunoprecipitation Assays forIdentification of the KOC1-KIF11 Binding Region

KOC1 and several of its domains (FIG. 3 a) were cloned into appropriatesites of N-terminal FLAG-tagged and C-terminal HA-tagged pCAGGS vector.COS-7 cells transfected only with an KOC1 deletion mutant, wereimmunoprecipitated with anti-HA agarose (SIGMA). Endogenous KIF11 bandswere detected with affinity-purified anti-KIF11 antibody by westernblotting.

TABLE 3 Primer sequence for constraction of deletion mutant by RT-PCRSEQ SEQ F ID NO. R ID NO. full 5′-ATGAACAAACTGTATATCGG-3′ 695′-CTTCCGTCTTGACTGAGG-3′ 70 length KOC1 5′-ATGAACAAACTGTATATCGG-3′ 715′-ATGAGCTTCAAGTTTCACC-3′ 72 DEL1 KOC1 5′-ATGAACAAACTGTATATCGG-3′ 735′-CTCCGTTTCTGATTGCTC-3′ 74 DEL2 KOC1 5′-ATGAACAAACTGTATATCGG-3′ 755′-AGGCAAATCACATGGTTTCTG-3′ 76 DEL3 KOC1 5′-TTGCCTCTGCGCCTGCTG-3′ 775′-CTTCCGTCTTGACTGAGG-3′ 78 DEL4 KOC1 5′-TTGCCTCTGCGCCTGCTG-3′ 795′-CTCCGTTTCTGATTGCTC-3′ 80 DEL5RNA-immunoprecipitation and cDNA Microarray Screening for Identificationof KOC1-Associated mRNAs

We adopted the RNA immunoprecipitation protocol of Niranjanakumari etal. (Niranjanakumari, S. et al. Methods 26, 182-190 (2002)) to analyzeRNA-protein interactions involving KOC1 in vivo. Immunoprecipitated RNAswere isolated from five lung-cancer cell lines (A549, LC319, PC14,RERF-LC-AI, and SK-MES-1). A 2.5-μg aliquot of T7-based amplified RNAs(aRNAs) from each immunoprecipiated RNA (IP-RNA) and from the total RNA(control) were reversely transcribed in the presence of Cy5-dCTP andCy3-dCTP respectively as described previously (Kikuchi, T. et al.Oncogene 22, 2192-2205 (2003)), for hybridization to a cDNA microarrayrepresenting 32,256 genes (IP-microarray analysis). To confirm thebinding to KOC1 of the mRNAs identified by IP-microarray analysis, wecarried out RT-PCR experiments using gene-specific primers and RNAs fromNSCLC cell extracts immunoprecipitated with anti-KOC1 antibody(IP-RT-PCR). To confirm the region of KOC1 required for binding to theKOC1-associated mRNAs, we also carried out northwestern blot analysis asbelow and IP-RT-PCR of KOC1-associated mRNAs from theseimmunoprecipitated extracts transfected with various KOC1 deletionmutants.

TABLE 4 Primer sequence for IP-RT-PCR SEQ SEQ F ID NO. R ID NO. CCT25′-TTATCCTGAACAGCTCTTTGGTG-3′ 81 5′-AAGCGAAGGTCAGCTAAATATCC-3′ 82 SBP25′-CTTTCTGAGCACACTACGGATCT-3′ 83 5′-AAGCCCTCTTACTTACAGGGAAA-3′ 84SLC25A3 5′-GGTTCCCCTGGATTTAGTGAA-3′ 85 5′-CAACAGTAAATCTGAAACTCTTGCC-3′86 RAB35 5′-GACAAAGGTAGCAAGAGGATTTC-3′ 87 5′-CTGGTGTTAAACTCGGTTCTTC-3′88 PSMB7 5′-CTAGTGAGTGAGGCTATTGCAGC-3′ 89 5′-GTCTCTTCTAGCACCTCAATCTCC-3′90 GL 5′-ATCTGACTTTCTGTCCACTGCAT-3′ 91 5′-TAATTCAGCATAAGCCAAAGCC-3′ 92PKP4 5′-ACACAGTATGGACTGAAATCGAC-3′ 93 5′-CACCTCAATCTGAACAAGGTTAG-3′ 94WINS1 5′-GGCCTCTCAAAGTCTGGTAGATT-3′ 95 5′-ATATTCCCACTTCAGAGACGACA-3′ 96

Northwestern Blot Analysis

Immunoprecipitated extracts from cells transfected with the KOC1deletion mutants (μM) were boiled in 2×SDS-sample buffer,electrophoresed through 10-20% gradient polyacrylamide gels (BIO-RAD)and transferred to a polyvinylidene difluoride membrane (Hybond-P). Themembrane was then blocked for 1 hour at room temperature in blockingbuffer (10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 mg/ml yeast tRNA), andwashed twice with 50 ml of 10 mM Tris-HCl (pH 7.8) for 5 min andincubated with DIG-labeled RNA probe in 5 ml of NWB buffer (10 mMTris-HCl (pH 7.8), 1 mM EDTA, 50 mM NaCl, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA) for 2 hours at room temperature. The membranewas washed four times with NWB buffer and the RNA probe bound to theproteins was then detected using DIG nucleic acid detection kit (Roche)according to the supplier's protocol.

Living-Cell Imaging of KOC1 and KIF11 Proteins and KOC1-Associated RAB35mRNA

Plasmids expressing ECFP-fused KOC1 (ECFP-KOC1) protein were preparedusing pECFP-N1 vectors (BD Biosciences Clontech). Plasmids expressingEYFP-fused KIF11 (EYFP-KIF11) protein were also prepared, using pEYFP-N1vectors (BD Biosciences Clontech). Time-lapse images of COS-7 cellstransfected with plasmids expressing ECFP-KOC1 or EYFP-KIF11 proteinswere captured for 5-15 hours by the Live Cell Imaging System (PowerIX81, OLYMPUS) and a confocal microscope (TCS SP2-AOBS, LeicaMicrosystems; FV1000 FLUOVIEW, OLYMPUS).

In vitro transcription of linearized plasmids carrying the full-lengthcDNA sequence of an KOC1-associated gene, RAB35, was performed usingDAVIS Lab's protocol (found on the World Wide Web at ed.ac.uk/˜ilan). Togenerate fluorescent riboprobes for in vivo co-localization with KOC1,the plasmids were transcribed using the mCAP RNA capping kit(Stratagene) in the presence of Alexa Fluor 546-labeled UTP (MolecularProbes). We constructed plasmids expressing EGFP-fused KOC1 (EGFP-KOC1)protein were prepared using pEGFP-N1 vectors (BD Biosciences Clontech).For live-cell imaging of co-localized EGFP-KOC1 and Alexa Fluor546-labeled RAB35 mRNA, COS-7 cells that had been transfected initiallywith pEGFP-KOC1 were additionally transfected 36 hours later with AlexaFluor 546-labeled RAB35 mRNA (3 μg per 3.5-cm culture dish) in thepresence of RNase Inhibitor (TAKARA). The plasmid-DNA and RNA sampleswere transfected using Lipofectamine 2000 (Invitrogen) according to themanufacturer's protocols. The cells were washed twice with PBS, andfresh medium was added 90 min after transfection with the labeled mRNA.The cells were allowed to recover in the incubator (37° C., 5% CO₂) for30 min before live-cell imaging for 3-6 hours with a confocal microscope(FV1000 FLUOVIEW, OLYMPUS). To investigate the specific transport ofmRNAs by KOC1-RNP complex from one cell to another cell, we prepared twodifferent COS-7-derived cells; the COS-7 cells transfected withpEGFP-KOC1 and Alexa Fluor 546-labeled RAB35 mRNA and the other,parental COS-7 cells simply labeled with CellTracker (Molecular Probes)according to the supplier's protocols. These two cell populations weremixed and co-cultured for 12 hours before live-cell imaging withconfocal microscope for 6 hours.

To investigate the translation of the mRNA transported by KOC1-RNPcomplex in the recipient cells, we prepared two types of COS-7-derivedcell; one type was COS-7 cells co-transfected with pCAGGS-FLAGtagged-KOC1 and -KIF11. After 24 hours culture, plasmid containingEGFP-fused RAB35 full length mRNA were re-transfected into these cells.The other type was COS-7 cells simply labeled with CellTracker (blue).These two cell-types were mixed and co-cultured for 24 hours beforelive-cell imaging with video microscope for 12 hours. Synthesis ofEGFP-tagged RAB35 mRNAs and corresponding proteins in theCellTracker-stained recipient cells (blue) as well as on the ultrafinestructure between the two cells was examined by in situ hybridizationand time-lapse video microscopy.

Fluorescent In Situ Hybridization

We carried out in situ hybridization with DIG-labeled probescomplementary to RAB35 or EGFP mRNA at 60° C. for 16 hours. The DIGlabel was detected using NBT-BCIP, an alkaline phosphatase colorsubstrate. Cells were washed, mounted and visualized on lightmicroscope. Fixed cells were hybridized with a mixture of DIG-labeledcomplementary to RAB35 mRNA for 16 hours in 50% formamide/2×SSC at 42°C. Cells were washed, mounted and visualized on confocal microscope.

(5) RNA Interference Assay

To prepare plasmid vector expressing short interfering RNA (siRNA), weamplified the genomic fragment of H1RNA gene containing its promoterregion by PCR using a set of primers, 5′-TGGTAGCCAAGTGCAGGTTATA-3′ (SEQID No: 44), and 5′-CCAAAGGGTTTCTGCAGTTTCA-3′ (SEQ ID No: 45) and humanplacental DNA as a template. The product was purified and cloned intopCR2.0 plasmid vector using a TA cloning kit according to the supplier'sprotocol (Invitrogen). The BamHI and XhoI fragment containing H1RNA wasinto pcDNA3.1(+) between nucleotides 1257 and 56, and the fragment wasamplified by PCR using

5′-TGCGGATCCAGAGCAGATTGTACTGAGAGT-3′ (SEQ ID No: 46) and

5′-CTCTATCTCGAGTGAGGCGGAAAGAACCA-3′ (SEQ ID No: 47),

The ligated DNA became the template for PCR amplification with primers,

5′-TTTAAGCTTGAAGACCATTTTTGGAAAAAAAAAAAC-3′ (SEQ ID No: 48) and

5′-TTTAAGCTTGAAGACATGGGAAAGAGTGGTCTCA-3′ (SEQ ID No: 49).

The product was digested with HindIII, and subsequently self-ligated toproduce psiH1BX3.0 vector plasmid having a nucleotide sequence shown inSEQ ID NO: 50.

The DNA fragment encoding siRNA was inserted into the GAP at nucleotide489-492 as indicated (−) in the following plasmid sequence (SEQ ID NO:50).

GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGGCTTGGTAGCCAAGTGCAGGTTATAGGGAGCTGAAGGGAAGGGGGTCACAGTAGGTGGCATCGTTCCTTTCTGACTGCCCGCCCCCCGCATGCCGTCCCGCGATATTGAGCTCCGAACCTCTCGCCCTGCCGCCGCCGGTGCTCCGTCGCCGCCGCGCCGCCATGGAATTCGAACGCTGACGTCATCAACCCGCTCCAAGGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTGCGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTGCAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTCTTTCCC----TTTTTGGGAAAAAAAAAAAAAAAAAAAAAACGAAACCGGGCCGGGCGCGGTGGTTCACGCCTATAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACAAGGTCAGGAGGTCGAGACCATCCAGGCTAACACGGTGAAACCCCCCCCCATCTCTACTAAAAAAAAAAAATACAAAAAATTAGCCATTAGCCGGGCGTGGTGGCGGGCGCCTATAATCCCAGCTACTTGGGAGGCTGAAGCAGAATGGCGTGAACCCGGGAGGCGGACGTTGCAGTGAGCCGAGATCGCGCCGACTGCATTCCAGCCTGGGCGACAGAGCGAGTCTCAAAAAAAAAACCGAGTGGAATGTGAAAAGCTCCGTGAAACTGCAGAAACCCAAGCCGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGT GCCACCTGACGTC

Using 30 μl of Lipofectamine 2000 (Invitrogen), 10 μg ofsiRNA-expression vector were transfected into NSCLC cell lines, A549 andLC319, both endogenously over-expressing KOC1, KIF11, NMU, GHSR1b,NTSR1, RAB35, and FOXM1. More than 90% of the transfected cellsexpressed the synthetic siRNAs, and endogenous expression of targetgenes (KIF11, GHSR1b, NTSR1, RAB35, or FOXM1) in these cells waseffectively suppressed. The transfected cells were cultured for fivedays in the presence of appropriate concentrations of geneticin (G418),and then, cell numbers and viability were measured by Giemsa stainingand triplicate MTT assays. The target sequences of the syntheticoligonucleotides for RNAi were as follows: control 1 (EGFP: enhancedgreen fluorescent protein (EGFP) gene, a mutant of Aequorea victoriaEGFP), 5′-GAAGCAGCACGACTTCTTC-3′ (SEQ. ID. NO. 29); control 2(Luciferase: Photinus pyralis luciferase gene),5′-CGTACGCGGAATACTTCGA-3′ (SEQ. ID. NO. 30); control 3 (Scramble:chloroplast Euglena gracilis gene coding for 5S and 16S rRNAs),5′-GCGCGCTTTGTAGGATTCG-3′ (SEQ. ID. NO. 31); siRNA-KIF11-1 (#1),5′-GTTAGTGTACGAACTGGAG-3′ (SEQ. ID. NO. 32); siRNA-KIF11-2 (#2),5′-GTGTCTCTGTTGGAGATCT-3′ (SEQ. ID. NO. 33); siRNA-KIF11-3 (#3),5′-GAAGGCAGTTGACCAACAC-3′ (SEQ. ID. NO. 34); siRNA-GHSR-1 (si-GHSR-1),5′-CCTCTACCTGTCCAGCATG-3′ (SEQ. ID. NO. 35); siRNA-NTSR1-1 (si-NTSR1-1),5′-GTTCATCAGCGCCATCTGG-3′ (SEQ. ID. NO. 36); siRNA-NTSR1-2 (si-NTSR1-2),5′-GGTCGTCATACAGGTCAAC-3′ (SEQ. ID. NO. 37), siRNA-RAB35 (si-RAB35),5′-GAGATGTTCAACTGCATCA-3′ (SEQ. ID. NO. 114), siRNA-FOXM1 (si-FOXM1),5′-GCAGCAGAAACGACCGAAT-3′ (SEQ. ID. NO. 108).

The oligonucleotides used for these siRNAs are shown below. Eachconstructs were prepared by cloning the following double-strandedoligonucleotide into the BbsI site in the psiH1BX3.0 vector. Thecorresponding nucleotide position relative to the KIF11, GHSR1b, NTSR1,RAB35 and FOXM1 nucleic acid sequence of SEQ ID NOs:1, 3, 5, 112, and106 are listed for each oligonucleotide sequence. Each oligonucleotideis a combination of a sense nucleotide sequence and an antisensenucleotide sequence of the target sequence of KIF11, GHSR1b, NTSR1,RAB35 and FOXM1. The nucleotide sequences of the hairpin loop structureof each siRNAs are also shown bellow. (endonuclease recognition sitesare eliminated from each hairpin loop structure sequence).

KIF11 si1 288-306(for the target sequence of gttagtgtac gaactggag/SEQ ID NO: 32)(insert F)Tccc gttagtgtacgaactggag ttcaagaga ctccagttcgtacactaac/SEQ ID NO: 51(insert R)Aaaa gttagtgtacgaactggag tctcttgaa ctccagttcgtacactaac/SEQ ID NO: 52(hairpin)gttagtgtacgaactggag ttcaagaga ctccagttcgtacactaac/SEQ ID NO: 53 KIF11si2 612-630(for the target sequence of gtgtctctgt tggagatct/SEQ ID NO: 33)(insert F)Tccc gtgtctctgt tggagatct ttcaagaga agatctccaacagagacac/SEQ ID NO: 54(insert R)Aaaa gtgtctctgt tggagatct tctcttgaa agatctccaacagagacac/SEQ ID NO: 55(hairpin)gtgtctctgt tggagatct ttcaagaga agatctccaacagagacac/SEQ ID NO: 56 KIF11si3 1700-1718(for the target sequence of gaaggcagtt gaccaacac/SEQ ID NO: 34)(insert F)Tccc gaaggcagtt gaccaacac ttcaagaga gtgttggtcaactgccttc/SEQ ID NO: 57(insert R)Aaaa gaaggcagtt gaccaacac tctcttgaa gtgttggtcaactgccttc/SEQ ID NO: 58(hairpin)gaaggcagtt gaccaacac ttcaagaga gtgttggtcaactgccttc/SEQ ID NO: 59 GHSR1bsi1 237-255(for the target sequence of cctctacctg tccagcatg/SEQ ID NO: 35)(insert F)Tccc cctctacctg tccagcatg ttcaagaga catgctggacaggtagagg/SEQ ID NO: 60(insert R)Aaaa cctctacctg tccagcatg tctcttgaa catgctggacaggtagagg/SEQ ID NO: 61(hairpin)cctctacctg tccagcatg ttcaagaga catgctggacaggtagagg/SEQ ID NO: 62 NTSR1si1 933-951(for the target sequence of gttcatcagc gccatctgg/SEQ ID NO: 36)(insert F)Tccc gttcatcagc gccatctgg ttcaagaga ccagatggcgctgatgaac/SEQ ID NO: 63(insert R)Aaaa gttcatcagc gccatctgg tctcttgaa ccagatggcgctgatgaac/SEQ ID NO: 64(hairpin)gttcatcagc gccatctgg ttcaagaga ccagatggcgctgatgaac/SEQ ID NO: 65 NTSR1si2 1074-1092(for the target sequence of ggtcgtcata caggtcaac/SEQ ID NO: 37)(insert F)Tccc ggtcgtcata caggtcaac ttcaagaga gttgacctgtatgacgacc/SEQ ID NO: 66(insert R)Aaaa ggtcgtcata caggtcaac tctcttgaa gttgacctgtatgacgacc/SEQ ID NO: 67(hairpin)ggtcgtcata caggtcaac ttcaagaga gttgacctgtatgacgacc/SEQ ID NO: 68 RAB35si 620-638(for the target sequence of gagatgttca actgcatca/SEQ ID NO: 114)(insert F)Tccc gagatgttca actgcatca ttcaagaga tgatgcagt tgaacatctc/SEQ ID NO: 115(insert R)Aaaa gagatgttca actgcatca tctcttgaa tgatgcagt tgaacatctc/SEQ ID NO: 116(hairpin)gagatgttca actgcatca ttcaagaga tgatgcagt tgaacatctc/SEQ ID NO: 117 FOXM1si 1240-1258(for the target sequence of gcagcagaaacgaccgaat/SEQ ID NO: 108)(insert F)Tccc gcagcagaaa cgaccgaat ttcaagaga attcggtcg tttctgctgc/SEQ ID NO: 109(insert R)Aaaa gcagcagaaa cgaccgaat tctcttgaa attcggtcg tttctgctgc/SEQ ID NO: 110(hairpin)gcagcagaaa cgaccgaat ttcaagaga attcggtcg tttctgctgc/SEQ ID NO: 111.

To validate RNAi system of the present invention, individual controlsiRNAs (EGFP, Luciferase, and Scramble) were initially confirmed usingsemiquantitative RT-PCR to decrease the expression of the correspondingtarget genes that had been transiently transfected into COS-7 cells.Down-regulation of KIF11, GHSR1b, NTSR1, RAB35 and FOXM1 expression bytheir respective siRNAs (si-KIF11-1, si-KIF11-2, si-KIF11-3, si-GHSR-1,si-NTSR1-1, si-NTSR1-2, si-RAB35 and si-FOXM1), but not by controls, wasconfirmed with semiquantitative RT-PCR in the cell lines used for thisassay.

Dominant-Negative Assays

We performed dominant-negative assays using the KOC1 deletion mutants toinvestigate the functional role of the KOC1-KIF11 complex in growth orsurvival of lung-cancer cells. The KOC1DEL3 and KOC1DEL2 construct (FIG.3 a; 10 μg), mock plasmid (10 μg), or plasmid mixtures of bothconstructs in the final dose of 10-μg DNA (KOC1DEL3 or KOC1DEL2 vs mock(μg), 7.5:2.5; 5:5; or 2.5:7.5, individually) were transfected intoLC319 cells. The transfected cells were cultured for 7 days in thepresence of G418 and their viability was measured by triplicate MTTassays.

(6) Co-Immunoprecipitation and MALDI-TOF Mass Spectrometry

Human lung cancer cell line LC319 cells were transfected withbilateral-tagged pCAGGS-n3FH (NH2-terminal FLAG, COOH-terminal HA)-KOC1expression vector or empty vector (mock transfection). Cells wereextracted in IP-buffer (0.5% NP-40, 50 mM Tris-HCl, 150 mM NaCl, andprotease inhibitor) for 30 min on ice. Extracts were centrifuged at14,000 rpm for 15 min, and supernatants were subjected toimmunoprecipitation using anti-Flag M2 agarose (Sigma-Aldrich) andanti-HA beads (Sigma-Aldrich) for 1-2 hours. The beads were washed sixtimes with IP-buffer, and protein was eluted by boiling the beads inLaemmli sample buffer after removing the final wash fraction. The elutedprotein was resolved by SDS-PAGE and stained with silver staining. A 125kDa-band was extracted by gel extraction, and used for massspectrometric sequencing using MALDI-TOF mass spectrometry. Thisanalysis identified KIF11 as the 125 kDa product.

To confirm the interaction between KOC1 and KIF11, A549 cells weretransiently co-transfected with Flag-tagged KIF11 and myc-tagged KOC1and the cells were immunoprecipitated with anti-Flag M2 agarose.Subsequently, the cells were immunoblotted with anti-myc antibody (9E10;Santa Cruz). Next, using the same combination of vectors and cells, thecells were immunoprecipitated with anti-myc agarose (SIGMA) andimmunoblotted with anti-Flag M2 antibody (Sigma-Aldrich).

To further confirm this interaction, A549 cells were transientlyco-transfected with Flag-tagged KIF11 and myc-tagged KOC1, andco-localization of FITC-labeled KIF11 and rhodamine-labeled KOC1 mainlyin the cytoplasm was detected by immunocytochemical staining usingFITC-labeled anti-FLAG antibody and rhodamine-labeled anti-myc antibody,as described below.

(7) Immunocytochemistry

A549 cells grown on coverslips were cultured for 24 hours after passage,and were co-transfected with Flag-tagged KIF11 and myc-tagged KOC1.After 24-hours incubation, the cells were fixed with acetone/methanol(1:1) for 5 min on ice, blocked in CAS BLOCK (ZYMED) for 7 min at RT,and then incubated with rabbit anti-Flag polyclonal antibody (SIGMA) for1 hour at RT. The fixed cells were washed 3 times with PBS, reacted withanti-rabbit IgG-FITC for 1 hour at RT. Then the cells were blockedagain, and incubated with anti-myc antibody (9E10; Santa Cruz) for 1hour at RT. Finally anti-mouse IgG-rhodamin was applied to the cells for1 hour at RT. The cells were viewed on a Leica TCS SP2-AOBS confocalmicroscope.

Immunohistochemistry and Tissue-Microarray Analysis

Tumor-tissue microarrays using formalin-fixed NSCLCs were constructed aspublished elsewhere (Kononen, J. et al., Nat. Med. 4, 844-847 (1998);Sauter, G. et al., Nat. Rev. Drug Discov. 2, 962-972 (2003)). KOC1 andKIF11 positivity were assessed semi-quantitatively as absent or positiveaccording to staining intensity, by three independent investigators withno prior knowledge of clinical follow-up data.

(8) Ligand-Receptor Binding Assay

To identify direct binding of NMU-25 to its candidate receptors, GHSR1a,GHSR1b and NTSR1, the following experiments were performed. The entirecoding region of each receptor gene was amplified by RT-PCR usingprimers

GHSR1a (5′-GGAATTCCATGTGGAACGCGACGCCCAGCGAA-3′ (SEQ. ID. NO. 38) and5′-CGCGGATCCGCGTGTATTAATACTAGATTCTGTCCAGGCC-3′ (SEQ. ID. NO .39)),GHSR1b (5′-GGAATTCCATGTGGAACGCGACGCCCAGCGAA-3′ (SEQ. ID. NO. 40) and5′-CGCGGATCCGCGGAGAGAAGGGAGAAGGCACAGGGA-3′ (SEQ .ID .NO .41)), and NTSR1(5′-GGAATTCCATGCGCCTCAACAGCTCCGCGCCGGGAA-3′ (SEQ. ID. NO. 42) and5′-CGCGGATCCGCGGTACAGCGTCTCGCGGGTGGCATTGCT-3′ (SEQ. ID. NO. 43)).

The products were digested with EcoRI and BamHI and cloned intoappropriate sites of p3XFLAG-CMV10 vector (Sigma-Aldrich Co.). COS-7cells were transfected with GHSR1b or NTSR1 expression plasmids usingFuGENE6, as described above. Transfected COS-7 cells were cultured with0.5 μM rhodamine-labeled NMU-25 peptide (NMU-25-rhodamine: PhoenixPharmaceuticals. Inc.) for 12 hours, washed five times in PBS(−), andfixed in 4% paraformaldehyde solution for 60 min at room temperature.Then the cells were incubated with antibodies to FLAG-tagged GHSR1a,GHSR1b, or NTSR1 proteins (Sigma-Aldrich Co.), stained with a goatanti-mouse secondary antibody conjugated to FITC (Cappel) and viewedunder laser-confocal microscopy (TCS SP2 AOBS: Leica Microsystems). Inaddition, three different negative controls were prepared for thisassay: 1) non-transfected COS-7 cells without addition ofNMU-25-rhodamine; 2) non-transfected COS-7 cells treated withNMU-25-rhodamine; and 3) COS-7 cells transfected with GHSR1a, GHSR1, orNTSR1 without NMU-25-rhodamine. COS-7 cells transfected with a known NMUreceptor (NMU1R) served as a positive control for the assay.

To confirm the binding of NMU-25 to the candidate receptors,flow-cytometric analysis was performed using the same series of COS-7cells. Specifically, COS-7 cells were plated at a density of 1×105cells/100-mm dish and transfected with either GHSR1b, NTSR1, or NMU1Rexpression vectors; 24 hours after transfection, cells were incubatedwith 0.5 μM NMU-25-rhodamine for 12 hours, washed, trypsinized,collected in PBS, and washed once more in PBS. The population of cellsbinding to rhodamine-labeled NMU-25 was determined by flow cytometry.

To further confirm binding of NMU-25 to the endogenous candidatereceptors on the NSCLC cells, we performed receptor-ligand binding assayusing the LC319 and PC-14 cells. Briefly, these cells trypsinized wereseeded onto 96-well black-wall, clear-bottom microtiter plates 24 hoursprior to the assay. The medium was removed and the cells were incubatedwith Cy5-NMU-25 with a 10-fold excess of unlabeled competitor. The platewas incubated in the dark for 24 hours at 37° C. and was scanned on the8200 Cellular Detection System (Applied Biosystems). 8200 AnalysisSoftware creates a digitized gray scale image, quantitates the amount offluorescence bound on the surface of each cell, and enumerates thefluorescent cells.

Measurement of cAMP Levels

Trypsinized LC319 cells were seeded onto 96-well microtiter plate(5.0×10⁴ cells) and cultured in 10% FCS (+) RPMI-1640 medium for 24hours, and then medium was changed to serum free RPMI-1640 medium/1 mMIBMX (isobutylmethylxanthine) for 20 min prior to assay. Cells wereincubated with NMU-25 peptides for 20 min prior to measuring the cAMPlevel using the cAMP EIA System (Amersham Biosystems).

Intracellular Ca²⁺ Mobilization Assay

Trypsinized LC319 cells were seeded onto poly-D-lysine coated 384-wellblack-wall, clear-bottom microtiter plate (1.0×10⁴ cells/ml) 24 hoursprior to assay. Cells were loaded for 1 hour with 1 mM Fluo-4-AMfluorescent indicator dye in assay buffer (Hank's balanced saltssolution, 20 mM HEPES, 2.5 mM probenecid), washed three times with assaybuffer, and then returned to the incubator for 10 min before assay on afluorometric imaging plate reader (FLIPR, Molecular Devices). Maximumchange in fluorescence over base line was used to determine the responseof the cells to the NMU-25 peptides stimulation.

Identification of Downstream Genes of NMU by cDNA Microarray

LC319 cells were transfected with either siRNA against NMU (si-NMU) orLuciferase (control siRNA). mRNAs were extracted 12, 24, and 36 hoursafter transfection, labelled with Cy5 or Cy3 dye and subjected toco-hybridization onto cDNA microarray slides containing 32,256 genes asdescribed (Kakiuchi, S., et al., (2004). Hum. Mol. Genet. 13, 3029-3043,Ochi, K. et al., (2004). Int. J. Oncol. 24, 647-655.). Afternormalization of the data, genes with signals higher than the cut-offvalue were analyzed further. Genes whose intensity were significantlydecreased in accordance with the time-dependent reduction of NMUexpression were initially selected using SOM cluster analysis.Validation of candidate downstream genes of NMU was performed usingsemiquantitative RT-PCR experiments of the same mRNAs from LC319 cellsused for microarray hybridization, with gene-specific primers listedbelow.

FLJ42024 (5′-AAAAAGGGGATGCCTAGAACTC-3′ (SEQ. ID. NO. 118) and5′-CTTTCAGCACGTCAAGGACAT-3′ (SEQ. ID. NO. 119)), GCDH(5′-ACACCTACGAAGGTACACATGAC-3′ (SEQ. ID. NO. 120) and(5′-GCTATTTCAGGGTAAATGGAGTC-3′ (SEQ. ID. NO. 121)), CDK5RAP1(5′-CAGAGATGGAGGATGTCAATAAC-3′ (SEQ. ID. NO. 122) and(5′-CATAGCAGCTTTAAAGAGACACG-3′ (SEQ. ID. NO. 123)), LOC134145(5′-CCACCATAACAGTGGAGTGGG-3' (SEQ. ID. NO. 124)(5′-CAGTTACAGGTGTATGACTGGGAG-3′ (SEQ. ID. NO. 125)), NUP188(5′-CTGAATACAACTTCCTGTTTGCC-3′ (SEQ. ID. NO. 126) and(5′-GACCACAGAATTACCAAAACTGC-3′ (SEQ. ID. NO. 127)).

Expression of the candidate genes was additionally detected bysemiquantitative RT-PCR using mRNAs isolated at 72 and 96 hours fromLC319 cells treated with 1 μM NMU-25 or BSA at the time point of 0 and48 hours.

Results

(1) Identification of KIF11 as a Protein Interacting with KOC1

LC319 cells transfected with pCAGGS-n3FH-KOC1 vector were extracted andimmunoprecipitated with anti-Flag M2 monoclonal antibody, andsubsequently immunoprecipitated with anti-HA monoclonal antibody. Theprotein complex including KOC1 was stained with silver staining onSDS-PAGE gel. A 125 kDa band that was absent in mock transfection wasextracted and determined to be KIF11 (NM_(—)004523; SEQ. ID. NO. 1) byMass spectrometric sequencing.

(2) Confirmation of Interaction Between KOC1 and KIF11

The A549 cells co-transfected with Flag-tagged KIF11 and myc-taggedKOC1, the cells transfected with either KIF11 or KOC1, and thenon-transfected cells were immunoprecipitated with anti-Flag M2 agaroseand subsequently immunoblotted with anti-myc antibody. In contrast, thesame series of A549 cells were immunoprecipitated with anti-myc agaroseand immunoblotted with anti-Flag M2 antibody. A single band was foundonly when both constructs were co-transfected (FIG. 1 a).Immunocytochemistry showed that FLAG-tagged FITC-labeled KIF11co-localized in cytoplasm of A549 with myc-tagged rhomamine-labeled KOC1(FIG. 1 b).

Next, we confirmed by western blot analysis that anti-KOC1 antibody arespecific to KOC1 and do not cross-react with other homologous proteins,IMP-1 and IMP-2 using H520 cell lysate, which had been confirmed to benot expressed endogenous IMP-1, -2, and -3 (KOC1), but had beentransfected with HA-tagged IMP-1, -2, and -3 (KOC1) expression vector.Lysates of LC319 cells transfected with pCAGGS-FLAG-tagged-KOC1 vectoror mock vector (control) were extracted and immunoprecipitated withanti-FLAG M2 monoclonal antibody. The protein complex including KOC1 wasstained with SilverQuest (Invitrogen) on an SDS-PAGE gel. A 125-kDa bandwas detected specifically in immunoprecipitates from lysates of cellstransfected with KOC1 expressing plasmids, but not in control lysates(mock plasmids). Subsequent MALDI-TOF mass spectrometric analysisidentified this 125-kDa protein as KIF11, a member of the kinesinfamily. We confirmed direct interaction between endogenous KOC1 andKIF11 by immunoprecipitation experiments with extracts from A549 andLC319, using affinity-purified anti-KOC1 and anti-KIF11 polyclonalantibodies (FIG. 1 c).

(3) KIF11 Expression in NSCLC

Validation of KIF11 expression was performed in primary NSCLCs (clinicalsamples) and lung cancer cell lines. Increased KIF11 expression wasconfirmed in 12 of 16 NSCLC cases (5 of 8 ADCs and in 7 of 8 SCCs. Inaddition, up-regulation of KIF11 were observed in 14 of the 15 NSCLCcell lines.

We subsequently re-examined primary NSCLC tissues and lung-cancer celllines, and found increased KIF11 expression in 18 NSCLC clinical samplesas well as in all of the 20 NSCLC or SCLC cell lines examined byquantitative RT-PCR (FIG. 2 a,b). The mRNA levels of the KOC1 and KIF11genes relative to ACTB genes were significantly correlated (r=0.702,P=0.0029 by the Spearman rank correlation). These two genes werecoactivated in almost lung cancer cell lines (r=0.458, P=0.0359 by theSpearman rank correlation).

(4) KIF11 Expression in Normal Human Tissues

Northern blotting with KIF11 cDNA as a probe identified 4.5- and 5.5-kbtranscripts as very weak bands, only seen in placenta, testis, and bonemarrow, among the 23 normal human tissues examined. The reported cDNAsequence of KIF11 was considered to correspond to the larger transcript.To investigate the transcript corresponding to the smaller band, wereversely transcribed mRNAs isolated from tissues of the testis andNSCLC cell lines. We amplified the entire sequence of KIF11 cDNA by PCRusing four primer sets, but found no alternatively-spliced transcript inthese samples. Therefore, the smaller band may reflectcross-hybridization to the transcript of some related gene(s). Theexpression pattern of KIF11 in normal human tissues was significantlycorrelated with that of KOC1 (FIG. 2 c).

Identification of the KIF11-Binding Region in KOC1

To determine the specific domain of KOC1 required for interaction withKIF11, we transfected into COS-7 cells one of five deletion-constructsof KOC1 with NH₂ (N)-terminal FLAG- or COOH (C)-terminal HA-taggedsequences (KOC1DEL1-5; FIG. 3 a). Immunoprecipitation with monoclonalanti-HA indicated that the KOC1DEL4 and KOC1DEL5 constructs, which bothlacked two RNA-recognition motifs (RRMs), were unable to interact withendogenous KIF11, while all KOC1 constructs possessing the two RRMsretained binding affinity for KIF11 (FIG. 3 b).

Isolation of mRNAs Associated with the KOC1-KIF11 Complex UsingRNA-Immunoprecipitation and cDNA Microarray

KOC1 protein is known to exhibit multiple attachments to IGF2 leader-3mRNA, possibly through its two functional RRMs and four K-homologous(KH) domains (Nielsen, J. et al., Mol. Cell Biol. 19, 1262-1270(1999).). However, we did not detect expression of IGF2 mRNA in any ofthe lung-cancer cell lines or clinical NSCLC tissue samples we examined.Therefore, to elucidate the function of KOC1 in lung carcinogenesis, wesearched for mRNA(s) that would interact with KOC1 and might therebyplay important roles in growth and/or progression of lung cancer. Firstwe immunoprecipitated mRNAs using anti-KOC1 antibody and five NSCLC celllines (A549, LC319, PC14, RERF-LC-AI, and SK-MES-1). Then, Cy-5-labeledimmunoprecipitated RNAs (IP-mRNA) and Cy-3-labeled total RNAs isolatedfrom each matching cell line, were co-hybridized on human cDNAmicroarrays (IP-microarray). Among 32,256 genes screened, we identifieda total of 55 that were enriched in IP-mRNA compared with total RNA inat least four of the five NSCLC cell lines tested (see Table 2), andconfirmed enrichment of all those candidates by RT-PCR using theIP-mRNAs as templates (IP-RT-PCR). To examine the specificity ofRNA-immunoprecipitation, we performed RT-PCR experiments with beta-actin(ACTB) mRNA using IP-mRNA as template; no ACTB was precipitated byanti-KOC1 antibody. As background controls of RNA-immunoprecipitation,we precipitated mRNAs using normal rabbit IgG and five NSCLC cell lines,and confirmed that none of eight KOC1 associated mRNAs tested (CCT2,SBP2, SLC25A3, RAB35, PSMB7, GL, PKP4, and WINS1) was precipitated bynormal rabbit IgG. We also confirmed elevated expression of many of thecandidate genes in NSCLC samples by RT-PCR (data not shown). To examinewhether the KOC1-KIF11 complex formation requires the co-presence ofthese KOC1-associated mRNAs, we performed immunoprecipitationexperiments using cell lysates which were treated or untreated in vitrowith 30 units of RNase T1 (SIGMA), and found no difference in theinteraction of the two proteins in the presence or absence of mRNAs,suggesting that the KOC1-KIF11 complex formation is unlikely to requirethese specific mRNAs.

By pursuing that strategy we have been able to show that KOC1 and KIF11not only are co-over-expressed in the great majority of clinical NSCLCsamples and cell lines, but also that a complex formed by the productsof these genes is indispensable for growth and progression of NSCLCcells, by contributing to an intra- and inter-cellular mRNA-transportingsystem. Intracellular mRNA transport by RNA-binding proteins has beenreported in oocytes and developing embryos of Drosophila and Xenopus,and in somatic cells such as fibroblasts and neurons (King, M. L. etal., Bioessays 21, 546-557 (1999); Mowry, K. L. & Cote, C. A. Faseb. J.13, 435-445 (1999); Lasko, P., J. Cell Biol. 150, F51-56 (2000);Steward, O. Neuron 18, 9-12 (1997)) beta-actin mRNA is transported tothe leading lamellae of chicken-embryo fibroblasts (CEFs) and to thegrowth cones of developing neurons (Lawrence, J. B. & Singer, R. H. Cell45, 407-415 (1986); Bassell, G. J. et al., J. Neurosci. 18, 251-265(1998)). The localization of ACTB mRNA depends on the “zipcode”, acis-acting element in the 3′UTR of the mRNA (Kislauskis, E. H. et al.,J. Cell Biol. 123, 165-172 (1993)). The respective trans-acting factor,zipcode-binding protein 1 (ZBP1), was identified by affinitypurification with the zipcode of ACTB mRNA; (Ross, A. F. et al., Mol.Cell Biol. 17, 2158-2165 (1997)) homologues of ZBP1 have since beenidentified in a wide range of organisms including Xenopus, Drosophila,mouse, and human (Mueller-Pillasch, F. et al., Oncogene 14, 2729-2733(1997); Deshler, J. O. et al., Science 276, 1128-1131 (1997); Doyle, G.A. et al., Nucleic Acids Res. 26, 5036-5044 (1998)). ZBP1-like proteinscontain two RRMs in the N-terminal region and four hnRNP KH(ribonucleoprotein K-homology) domains at the C-terminal end. KOC1, oneof the IGF2 mRNA-binding proteins, is considered to be a member of theZBP1 family; it exhibits multiple attachments to IGF2 leader-3 mRNA(Nielsen, J. et al., Mol. Cell Biol. 19, 1262-1270 (1999)) and isover-expressed in several types of cancers (Mueller-Pillasch, F. et al.,Oncogene 14, 2729-2733 (1997); Zhang, J. Y. et al., Clin. Immunol. 100,149-156 (2001); Mueller, F. et al., Br. J. Cancer 88, 699-701 (2003);Wang, T. et al., Br. J. Cancer 88, 887-894 (2003)). However, since wefailed to detect expression of IGF2 leader-3 mRNA in most of the NSCLCcell lines or clinical samples we examined, we suspected that KOC1 couldmediate growth of lung-cancer cells through interaction with, andtransport of, other mRNA(s). When we undertook RNA-immunoprecipitationexperiments coupled with cDNA microarrays (IP-microarray), we identifieddozens of candidate mRNAs that were likely to be associated with KOC1 inNSCLC cells (see Table 2). That list included genes encoding proteinswith functions of cell-adhesion (PKP4, L1CAM1), cancer-cell progressionand invasion (IGFBP2), and binding of small GTPs (RAB35), (Papagerakis,S. et al., Hum. Pathol. 34, 565-572 (2003); Fogel, M. et al., CancerLett. 189, 237-247 (2003); Wang, H. et al., Cancer Res. 63, 4315-4321(2003); Zhao, H. et al., Biochem. Biophys. Res. Commun. 293, 1060-1065(2002)) and many of them were expressed at high levels in clinical NSCLCsamples (data not shown). Activation of a system that transports mRNAswhose products are associated with growth or movement of cells is veryinteresting, and further investigations along this line could lead to abetter understanding of pulmonary carcinogenesis.

TABLE 2 RANK⁽¹⁾ GENE ACCESSION A549 LC319 PC14 RERF SKMES1 SUM*  1LOC283050 AA843724 6.0 7.7 5.4 6.4 9.2 34.7  2 KIAA0169 R49113 6.2 8.94.9 5.7 6.8 32.5  3 CCT2 AF026166 4.1 8.0 5.4 5.7 9.1 32.3  4 LOH11CR2ANM_014622 9.1 5.6 5.8 3.9 4.5 28.8  5 SNTB2 AA625860 6.0 5.0 6.8 4.0 5.026.9  6 CFLAR U97074 5.3 5.7 3.8 4.3 5.9 25.0  7 SBP2 AF380995 5.0 6.13.9 2.9 6.1 24.0  8 LOC56267 AA420728 4.8 5.4 5.4 2.5 5.0 23.1  9SLC25A3 NM_002635 3.5 5.8 2.9 4.1 5.3 21.7 10 IFIT1 M24594 4.4 4.2 3.83.6 5.2 21.2 11 OSTalpha H79642 2.3 5.9 5.6 3.8 2.8 20.4 12 FILIP1XM_029179 10.3 2.3 2.1 2.7 2.5 19.9 13 ZNF415 AY283600 3.5 5.2 3.3 4.03.8 19.8 14 RAB35 BX344673; 3.0 4.1 4.2 4.0 4.6 19.8 NM_006861 15 APG-1AW966019 0.0 6.2 6.2 2.5 4.4 19.4 16 INPP4B AA759168 3.4 3.2 4.4 3.6 4.519.1 17 na AI160370 3.6 4.1 2.9 4.6 3.1 18.3 18 N33 NM_006765 4.5 0.04.0 4.7 5.0 18.1 19 RPS3A BX343424 1.6 4.5 3.2 3.1 4.3 16.8 20 PSMB7BM837906 4.0 4.2 2.2 3.1 2.9 16.5 21 GIT2 NM_057169 3.4 4.2 2.3 2.9 3.416.1 22 GL AJ420489 4.4 3.2 3.2 2.3 2.9 16.0 23 SOS2 XM_043720 2.1 3.52.5 2.3 4.7 15.1 24 L1CAM M77640 3.4 2.9 2.5 2.6 3.6 14.9 25 BRUNOL4BM671360 2.8 3.8 1.7 2.5 4.1 14.9 26 RRAGA U41654 2.9 4.3 2.4 2.4 2.814.8 27 IGFBP2 BC004312 3.9 3.7 2.0 2.5 2.6 14.8 28 SRPK1 BC038292 3.32.5 2.8 2.8 3.4 14.8 29 FLJ12649 R41135 1.2 2.8 2.6 3.4 4.5 14.4 30 AGLNM_000028 4.0 2.8 2.6 2.5 2.4 14.3 31 FLJ23468 BX355581 3.0 3.4 3.0 2.12.4 13.9 32 MGC4730 BM665147 2.6 2.8 2.6 2.3 3.1 13.4 33 GAB2 NM_0122963.7 3.4 1.3 2.8 2.2 13.4 34 USP15 AF106069 2.0 3.0 2.4 2.6 3.2 13.1 35KIAA0657 AB014557 0.0 4.7 2.2 3.0 3.1 13.1 36 C6orf134 AI146643 3.2 0.02.5 2.7 4.5 12.8 37 MSCP AK093931 2.5 3.0 4.2 3.0 0.0 12.7 38 ACAA2D16294 2.1 2.0 3.0 2.6 3.1 12.7 39 PKP4 AI681111 3.2 2.5 2.9 1.7 2.312.6 40 RGS5 BX537427 2.0 3.0 1.3 3.4 2.8 12.5 41 CYFIP1 BC005097 2.22.1 2.6 1.3 4.0 12.2 42 PLAGL2 AK026951 1.2 2.7 2.3 2.4 3.3 11.9 43 EHD4AW779971 2.7 2.3 2.3 1.9 2.6 11.9 44 KIAA1666 XM_300791 2.1 2.9 2.4 2.32.2 11.9 45 RAP80 BX537376 2.4 2.1 3.0 1.8 2.5 11.7 46 LOC118812BG537484 0.0 2.3 2.3 3.2 3.7 11.5 47 UTX AF000993 1.0 2.2 2.8 3.2 2.211.4 48 PCBP3 AK094301 2.4 2.9 1.3 2.3 2.5 11.4 49 AP3S2 BC002785 2.42.3 1.2 2.8 2.3 11.0 50 WINS1 AA741459 1.4 3.0 2.2 2.1 2.2 10.9 51 na⁽²⁾AF504647 0.8 2.0 2.1 2.1 3.6 10.7 52 LOC203859 AL832374 2.0 2.6 2.5 3.40.0 10.5 53 HNMT NM_006895 2.3 2.0 1.9 2.1 2.2 10.5 54 LOC282965XM_210833 1.1 2.8 2.0 2.2 2.0 10.1 55 PDK2 AK055119 1.0 2.4 2.2 2.4 2.010.0 N/C⁽³⁾ ACTB BC053988 0.0 0.0 0.0 0.0 0.0 0.0 ⁽¹⁾Probe sets areranked by the sum (*) of the fold change value (IP-mRNA/input RNA) ofall five cell lines. ⁽²⁾na: not annotated ⁽³⁾N/C: negative controlIdentification of the mRNA-Binding Region in KOC1

To determine the region of KOC1 that is required for binding toKOC1-associated mRNAs, we performed northwestern blot analysis usingimmunoprecipitated recombinant proteins of KOC1 deletion-mutantsexpressed in A549 cells (FIG. 4 a) and DIG-labeled RAB35 mRNA, which isone of the KOC1 associated mRNAs. The KOC1DEL3, which lacked four KHdomains, and KOC1DEL5, which lacked N-terminal two RRMs and C-terminaltwo KH domains, did not bind to the RAB35 mRNA. On the other hand, theKOC1DEL4, which is a construct with only the four KH domains and theKOC1DEL2, a construct without C-terminal two KH domains showed very weakbinding affinities for mRNAs compared to the full-length KOC1 construct(FIG. 4 b), suggesting the importance of two RRMs as well as ofC-terminal two KH domains for binding to KOC1-associated mRNAs.

We further expressed five of the KOC1 deletion-mutants in A549 cells andperformed immunoprecipitation experiments twice with the cell lysates,first with monoclonal anti-HA and then with monoclonal anti-FLAG M2antibody. Using IP-mRNA, we examined the ability of each deleted-proteinto bind to eight endogenous mRNAs (CCT2, SBP2, SLC25A3, RAB35, PSMB7,GL, PKP4, and WINS1) selected from the above list (see Table 2). Theresults were completely concordant to that of northwestern blotanalysis, independently confirming that both C-terminal two KH domainsand two RRMs in the N-terminal are indispensable for effective bindingof KOC1 to mRNAs (FIG. 4 c).

Microtubule Dependent Intra- and Inter-Cellular Transport of anKOC1-KIF11 Ribonucleoprotein Complex and KOC1-Associated mRNAs

To further investigate the functional roles of KOC1 and KIF11, weprepared plasmids designed to express ECFP-KOC1 (cyan) and EYFP-KIF11(yellow). We then transfected the two plasmids together into COS-7cells, and examined their localization using immunofluorescencevideo-microscopy and real-time confocal microscopy. Cells expressingboth KOC1 and KIF11 protruded into the processes, and then connectedwith adjacent cells (data not shown). A more detailed observation ofliving cells found that the KOC1 had formed a complex with KIF11(KOC1-KIF11 RNP complex; green particle) that was transported from onecell to another through an ultrafine structure connecting the two cells(FIG. 5 a). Movement of the KOC1-KIF11 complex appeared to beunidirectional from one cell to another.

Furthermore, to examine whether KOC1-KIF11 complex could specificallytransport KOC1-associated-mRNAs from one cell having a high level ofKOC1-RNP complex to another having a lower level of the complex, wemixed and co-cultured two different cell populations; one is COS-7 cellsthat had been transfected with pEGFP-KOC1 (green) as well as Alexa Fluor546-labeled full-length RAB35 mRNA (red), and the other is parentalCOS-7 cells simply labeled with CellTracker (blue). We observed that notonly KOC1 particles (green), but also RNP particles of KOC1-RAB35 mRNA(yellow) were transferred through the ultrafine structure from theformer cells to the latter ones (FIG. 5 b). Using in situ hybridizationon A549 cells in which both KOC1 and KIF11 were over-expressed, wefurther confirmed that the endogenous RAB35 mRNA (green) localized onthe ultrafine intercellular structures as well as in the cytoplasm (datanot shown).

We also investigated the endogenous location of KOC1 and KIF11 particleson the ultrafine structure of microtubules bridging individual A549cells by an immunocytochemical study, using affinity-purified anti-KOC1-or anti-KIF11 for primary antibody and Alexa594-labeled anti-rabbit IgGfor secondary antibody (Molecular Probe) and anti-alpha-tubulin-FITCmonoclonal antibody. A549 cells treated with 10 μM of the microtubuledisrupting agent nocodazole (SIGMA) for four hours showed collapse andaggregation of endogenous KOC1 and KIF11, along with thedepolymerization of microtubules in the cytoplasm. Moreover, no particlewas found on the residual structure between the cells. The resultsuggested the possibility of a microtubule-dependent transportingmechanism involving the KOC1-KIF11 complex. To further clarify thedetailed mechanism by which the KOC1-KIF11 complex transports mRNAs inNSCLC cells, we have searched for other component(s) that might beinteracting with KIF11. Immunoprecipitation with anti-KIF11 polyclonalantibody using a lysate of LC319 cells identified a 150-kDa protein,which was later determined to be a dynactin 1 (DCTN1; p150, gluedhomolog, Drosophila) by MALDI-TOF mass-spectrometric analysis. DCTN1 isthe largest subunit of DCTN, which binds to the cytoplasmicmotor-protein dynein and activates vesicle transport along microtubules(Holzbaur, E. L. & Tokito, M. K. Genomics 31, 398-399 (1996); Tokito, M.K. et al., Mol. Biol. Cell 7, 1167-1180 (1996)), or binds to KIF11 toprobably participate in centrosome separation (Blangy, A. et al., J.Biol. Chem. 272, 19418-19424 (1997)). We observed endogenousco-localization of KOC1/KIF11 and DCTN1 on the ultrafine structurebetween the individual A549 cells by immunocytochemistry, using thecombination of affinity-purified anti-KOC1- or anti-KIF11-polyclonalantibodies for primary antibody and Alexa488-labeled anti-rabbit IgG forsecondary antibody, and the combination of anti-DCTN1 monoclonalantibody (BD transduction Laboratories, #610473) for primary antibodyand anti-Alexa594-labeled anti-rabbit IgG for secondary antibody. And weconfirmed direct interaction between endogenous KIF11 and DCTN1 byimmunoprecipitation experiments with extracts from A549 and LC319 cells,using anti-KIF11 polyclonal antibody and anti-DCTN1 monoclonal antibody(BD transduction Laboratories, #610473) (FIG. 6 a).

To further demonstrate the KIF11-dependent intercellular transport ofmRNA, we examined the effect of monastrol, the cell-permeable inhibitorthat specifically inhibits the KIF11. Previous reports indicated thatmonastrol partially inhibits KIF11 ATPase activity through bindingdirectly to the motor domain (DeBonis, S. et al., Biochemistry 42,338-349 (2003); Kononen, J. et al., Nat. Med. 4, 844-847 (1998)).Treatment of A549 cells with 150 μM monastrol (SIGMA) for 24 hoursinduced the accumulation of endogenous KIF11 and exogenous EYFP-KIF11 atthe center of monoaster along microtubules and the cell cycle arrest inmitosis with monopolar spindles, which is called “monoastral spindle”.Treatment of A549 cells with 150 μM of monastrol for 24 hours inducedcell cycle arrest for mitotic cells with monopolar spindles that iscalled “monoastral spindle” and also caused accumulation of endogenousKIF11 at the center of monoaster along microtubules. On the other hand,most non-mitotic cells lost protrusion into the processes and then lostconnection to adjacent cells within 2-hour of the monastrol treatment.Further quantitative analysis by counting the number of intercellularultrafine structures (n=252 structures) with time-lapse video-microscopydemonstrated that more than a half of the cell-to-cell connections innon-mitotic cells tested disappeared by the one-hour monastroltreatment. However, six hours after the release of the cells from themonastrol exposure, the intercellular bridge formation wasre-constituted and cells at normal mitosis process was observed,indicating that KIF11 was indispensable for the formation of ultrafineintercellular structures (data not shown).

Some cells lost protrusion into the processes and then did not connectedwith adjacent cells. A more detailed observation of living cells foundthat no KOC1-KIF11 RNP complex (green particle) was transported from onecell to another through an ultrafine structure connecting the two cells,which subsequently disappeared during observation.

In this study we demonstrated endogenous interaction of KOC1, KIF11 andDCTN1 in human lung cancers, and revealed a possible role of thosecomplexes in transport of mRNAs from one cell to another. DCTN1, thelargest subunit of DCTN, binds to the cytoplasmic motor protein dyneinand activates vesicle transport along microtubules (Holzbaur, E. L. &Tokito, M. K. Genomics 31, 398-399 (1996)). Dynein-DCTN interaction isprobably a key component of the mechanism of axonal transport ofvesicles and organelles (Holzbaur, E. L. & Tokito, M. K. Genomics 31,398-399 (1996); Tokito, M. K. et al., Mol. Biol. Cell 7, 1167-1180(1996)). The binding of DCTN to dynein is reportedly critical forneuronal function, since antibodies that specifically disrupt thisbinding block vesicle motility along microtubules. In vitro interactionof DCTN1 and KIF11, and their co-localization during mitosis have beenobserved (Blangy, A. et al., J. Biol. Chem. 272, 19418-19424 (1997)),but no report has shown an intercellular transporting system involvingthis complex. Since in our experiments KIF11, a member of the kinesinfamily, was over-expressed in NSCLCs along with KOC1, we suggest thatdirect interaction of KOC1, KIF11, and DCTN1 could play a significantrole in establishing specific alignment of microtubules betweenlung-cancer cells.

Protein Synthesis by Transported KOC1-Associated mRNAs in the ReceivingCells

To elucidate whether the mRNA transport by KOC1-KIF11 RNP complex isphysiologically relevant (the recipient cell can synthesize the proteinby translating the mRNAs transported), we constructed an expressionvector of full length RAB35 mRNA, one of the binding targets of theKOC1/KIF11 complex, fused in frame to myc tagged and an EGFP proteinsequences. We then investigated whether this chimeric mRNA could betransportable from one cell to another and subsequently translated intothe protein production in the recipient cell. FLAG-tagged KOC1 and KIF11expressing-COST cells were transfected with constructs with these RAB35mRNA-expressing construct (cell A). Parental mRNA-recipient COS-7 cellswere simply stained with CellTracker (blue; cell B). These two cellpopulations were mixed together and co-cultured for 24 hours. We firstconfirmed the intercellular transportation of RAB35-EGFP mRNAs betweencells A and B by in situ hybridization using antisense EGFP as a probe;after co-culture of the cells for 24 hours, weak-staining of RAB35-EGFPmRNAs were detected in the CellTracker-stained cell B as well as on theultrafine structure between the two cell types (FIG. 7 a). We thenexamined a presence of the EGFP-fused RAB35 proteins in theCellTracker-stained B-type cells were found using immunocytochemistryand time-lapse video microscopy, respectively (FIGS. 7 b and 7 c).During these observations using time-lapse video microscopy, no visibleEGFP-protein particle was transported from the type-A to type-B cells,but the EGFP protein gradually appeared in the apparatus of cytoplasm,which seemed to be endoplasmic reticulum (ER) of in the type-B cells(FIG. 7 d). These results have indicated that KOC1 and KIF11 shouldfunctionally associate with a subset of mRNAs, which encode proteinspossibly inducing cell proliferation and/or adhesion, and that thepresence of KOC1 and KIF11 is indispensable to the cell-to-celltransportation. Although previous reports suggested that high KOC1levels might interfere with translation of bound mRNAs such as IGF2leader-3, our experiment of co-transfecting KOC1 and full-lengthRAB35-EGFP mRNA constructs together into COS-7 cells detected nodecrease of RAB35-EGFP-fused protein levels (FIG. 7 e).

Our experiments also revealed formation of protruding processesconnecting adjacent cells, and showed predominant co-distribution oftransfected RAB35 mRNAs and KOC1 protein on ultra-fine intercellularstructures in two lung-cancer cell lines (A549 and LC319) that expressedhigh levels of endogenous KOC1 and KIF11. On the other hand, we did notfind specific localization of transfected RAB35 mRNAs in NCI-H520 cells,which express KIF11 but not KOC1. That observation supported theimportance of co-activation of KOC1 and KIF11 for communication amongcancer cells. Among the known cell-to-cell communication systems inhuman cancers, formation of functional gap junctions between malignantglioma cells and vascular endothelial cells appears to influenceangiogenesis in the tumors (Zhang, W. et al., Cancer Res. 59, 1994-2003(1999); Zhang, W. et al., J. Neurosurg. 98, 846-853 (2003)). However, toour knowledge ours is the first report to describe inter-cellulartransport of mRNA by means of ribonucleoprotein particles combined withmotor proteins in mammalian somatic cells and to assess its biologicalsignificance for formation of an inter-cellular network critical forgrowth and survival of cancer cells.

(5) Inhibition of Growth of NSCLC Cells by siRNA Against KIF11

Transfection of either siRNA plasmids for KIF11 into A549 (FIG. 8 a) orLC319 (data not shown) cells suppressed mRNA expression of the KIF11 incomparison to cells containing any of the three control siRNAs and mocktransfection. In accordance with the reduced mRNA expression, A549 andLC319 cells showed significant decreases in cell viability and colonynumbers measured by MTT (FIG. 8 b) and colony-formation assays (data notshown). We also investigated the effect by siRNA against KIF11 onintercellular transport using time-lapse videoscopy. A similarphenomenon to monastrol treatment was observed; some cells reducedprotrusion into the processes and the disappearance of the ultrafinestructure connecting the two cells.

To investigate the functional significance of KOC1-KIF11 interaction forgrowth or survival of lung-cancer cells, a deletion fragment of KOC1containing the two RRMs, which was able to interact with KIF11(KOC1DEL3; FIG. 3 a, b) was examined for a dominant-negative function ofsuppressing direct interaction between endogenous KOC1 and KIF11. Wetransfected KOC1DEL3 and mock plasmid (control) into LC319 cells anddetected interaction of KOC1DEL3 with endogenous KIF11. We furtherverified that overexpression of the RRM domains reduce complex formationbetween KOC1 and KIF11 by immunoprecipitation (FIG. 9 a,b). Expectedly,transfection of that fragment resulted in significant dose-dependentdecreases in cell viability as measured by MTT assay (P<0.001, KOC1DEL3vs mock; FIG. 9 c). We also confirmed that transfection of constructcontaining only KH-domains control have no effect on proliferation.

Furthermore, to investigate the functional significance of KOC1-KIF11interaction for growth or survival of lung-cancer cells, a deletionfragment of KOC1, which lacked the C-terminal two KH-domainsindispensable for mRNA binding but was able to interact with KIF11(KOC1DEL2; FIG. 3 a, b), was examined for a dominant-negative functionof suppressing direct interaction between endogenous KOC1 and KIF11. Wetransfected KOC1DEL2 and mock plasmid (control) into A549 cells anddetected interaction of KOC1DEL2 with endogenous KIF11 (FIG. 9 d). Wefurther verified by immunoprecipitation that over-expression of theKOC1DEL2 reduced complex formation between endogenous KOC1 and KIF11(FIG. 9 e). Expectedly, transfection of the dominant-negative fragmentresulted in significant dose-dependent decreases in cell viability asmeasured by MTT assay (P=0.0006, KOC1DEL2 vs mock; FIG. 9 f).

We also examined some biological role(s) of these KIF11-transportingmRNAs in controlling the cell growth or survival of lung-cancer cells,we constructed plasmid to express siRNA against RAB35 (si-RAB35), whichwas identified as the KOC1-RNP complex-associated mRNAs. Transfection ofthe plasmids (si-RAB35) into A549 cells significantly suppressedexpression of endogenous RAB35 in comparison with the controls, andresulted in significant decreases in cell viability and colony numbersmeasured by MTT and colony-formation assays (FIG. 10 a,b).

Association of KOC1 and KIF11 Over-Expression with Poor Prognosis ofNSCLC Patients

We performed immunohistochemical analysis with anti-KOC1 and anti-KIF11polyclonal antibodies using tissue microarrays consisting of 265 NSCLCtissues (FIG. 11 a). Of the 265 cases, KOC1 staining was positive for172 (64.9%); 129 cases were positive for KIF11 (48.7%). The expressionpattern of KOC1 was significantly concordant with KIF11 expression inthese tumors (X²=60.8, P<0.0001). We then asked whether KOC1 and/orKIF11 over-expression could be associated with clinical outcome. Wefound that expression of KOC1 in NSCLCs was significantly associatedwith pT factor status (X²=23.1, P<0.0001) and with tumor-specific 5-yearsurvival (P=0.0115 by the Log-rank test) (FIG. 11 b, upper panel).Expression of KIF11 in NSCLCs was significantly associated with pTfactor (X²=15.0, P<0.0001), pN factor (X²=4.4, P=0.0356), and 5year-survival (P=0.0008 by the Log-rank test) (FIG. 11 b, lower panel).By univariate analysis pT, pN, gender, and KOC1/KIF11 expression wereeach significantly related to a poor tumor-specific survival among NSCLCpatients. Furthermore, KOC1 and KIF11 were determined to be independentprognostic factors by multivariate analysis using a Coxproportional-hazard model (P=0.0499 and P=0.0259, respectively).

(6) Screening of Candidate Receptors for NMU in NSCLC

Two known NMU receptors, NMU1R (FM3/GPR66) and NMU2R (FM4) playimportant roles in energy homeostasis (Fujii, R. et al., J. Biol. Chem.275: 21068-21074 (2000); Howard, A. D. et al., Nature 406: 70-74 (2000);Funes, S. et al., Peptides 23: 1607-1615 (2002)). NMU1R is present inmany peripheral human tissues (Fujii, R. et al., J. Biol. Chem. 275:21068-21074 (2000); Howard, A. D. et al., Nature 406: 70-74 (2000);Funes, S. et al., Peptides 23: 1607-1615 (2002)), but NMU2R is locatedonly in brain. To investigate whether NMU1R and NMU2R genes wereexpressed in NSCLCs, expression of these NMU receptors were analyzed innormal human brain and lung, in NSCLC cell lines, and in clinicaltissues by semiquantitative RT-PCR experiments. Neither NMU1R nor NMU2Rexpression was detected in any of the cell lines or clinical samplesexamined, although NMU1R was expressed in lung and NMU2R in brain (datanow shown), suggesting that NMU could be mediating growth of lung-cancercells through interaction with other receptor(s).

Since NMU2R and NMU1R were originally isolated as homologues of knownneuropeptide GPCRs, unidentified NMU receptor(s) were speculated to bemembers of the same family that would show some degree of homology toNMU1R/NMU2R. Hence, candidate NMU receptors were searched using theBLAST program. The results and their high expression levels in NSCLCs inthe expression profile data of the present inventors indicated GHSR1b(NM_(—)004122; SEQ ID NOs: 3 and 4) and NTSR1 (NM_(—)002531; SEQ ID NOs:5 and 6) to be good candidates. GHSR has two transcripts, types 1a and1b. The full-length human type 1a cDNA encodes a predicted polypeptideof 366 amino acids with seven transmembrane domains, a typical featureof G protein-coupled receptors. A single intron divides its open readingframe into two exons encoding transmembrane domains 1-5 and 6-7, thusplacing the GHSR1a into the intron-containing class of GPCRs. Type 1b isa non-spliced mRNA variant transcribed from a single exon that encodes apolypeptide of 289 amino acids with five transmembrane domains. Thesemiquantitative RT-PCR analysis using specific primers for each variantindicated that GHSR1a was not expressed in NSCLCs. On the other hand,GHSR1b and NTSR1 were expressed at a relatively high level in some NSCLCcell lines, but not at all in normal lung (FIG. 13 a). The GHSR1bproduct has 46% homology to NMU1R, and NTSR1 encodes 418 amino acidswith 47% homology to NMU1R.

(7) Identification of Candidate Receptors for NMU in NSCLC

To confirm direct interaction between NMU and GHSR1b/NTSR1, COS-7 cellswere transiently transfected with plasmids designed to expressFLAG-tagged GHSR1b or NTSR1, and cultured in the presence ofrhodamine-labeled NMU-25. Then the localization of FLAG-taggedGHSR1b/NTSR1 and NMU-25-rhodamine in the cells were examined usinganti-FLAG antibodies conjugated to FITC, and found that NMU-25 andeither of both receptors were located together on the cell membrane(FIG. 13 c). Co-localization of NMU-25 with these receptors was notobserved in control assays involving either of the following ligand/cellcombinations: 1) NMU-25-rhodamine incubated with COS-7 cells that werenot transfected with either of the receptor plasmids; 2) non-transfectedCOS-7 cells incubated without NMU-25-rhodamine; and 3) COS-7 cellstransfected with either of the receptor plasmids, but incubated withoutNMU-25-rhodamine. The result was confirmed by flow cytometry, whichrevealed binding of rhodamine-labeled NMU-25 to the surface of COS-7cells that expressed either of the two receptors (FIG. 13 d) and bindingof rhodamine-labeled NMU-25 to the surface of COS-7 cells in a dosedependent manner.

(8) GHSR1b Expression in Normal Human Tissues

As the expression of GHSR1b in normal human tissues was not preciselyreported at the time, the distribution of GHSR1b was determined usinghuman multiple tissue Northern-blot. Northern blotting with GHSR1b cDNAas a probe identified a 0.9-kb transcript as a very weak signal band incomparison with a 1.1-kb transcript GHSR1a, seen in the heart, liver,skeletal muscle, pancreas, and stomach, among the 23 normal humantissues examined (FIG. 13 b).

To further confirm binding of NMU-25 to the endogenous GHSR1b and NTSR1on the NSCLC cells, we performed receptor-ligand binding assay using theLC319 and PC-14 cells treated with NMU-25. We detected binding ofCy5-labeled NMU-25 to the surface of these two cell lines that expressedboth of the two receptors, but scarcely expressed NMU1R/NMU2R (FIG. 13e).

Biologically active ligands for GPCRs have been reported to bindspecifically to their cognate receptors and cause an increase insecond-messengers such as intracellular-Ca²⁺ and cAMP levels. Wetherefore determined the ability of NMU to induce thesesecond-messengers in LC319 cells through its interaction withGHSR1b/NTSR1. cAMP production, but not Ca²⁺ flux in LC319 cells, whichexpress both GHSR1b and NTSR1 was observed in a NMU-25 dose dependentmanner, when the cells were cultured in the presence of NMU-25 at finalconcentrations of 3-100 μM in the culture media. The results demonstratethat NSCLC cells express functional GHSR1b/NTSR1 (FIG. 13 f left panel).This effect was confirmed to be NMU-25 specific by adding other reportedligands for GHSR1b/NTSR1, GHRL or NTS (FIG. 13 f right panel). Inaddition, GHRL and NTS caused the mobilization response of intracellularcalcium in LC319 cells (data not shown), suggesting a variety offunction for the poorly understood for GHSR1b and/or NTSR1.

(9) Inhibition of Growth of NSCLC Cells by siRNA Against GHSR/NTSR1

Furthermore, the biological significance of the NMU-receptor interactionin pulmonary carcinogenesis was examined using plasmids designed toexpress siRNA against GHSR or NTSR1 (si-GHSR-1, si-NTSR1-1, andsi-NTSR1-2). Transfection of either of these plasmids into A549 or LC319cells suppressed expression of the endogenous receptor in comparison tocells containing any of the three control siRNAs (FIG. 14 a). Inaccordance with the reduced expression of the receptors, A549 and LC319cells showed significant decreases in cell viability (FIG. 14 b) andnumbers of colonies (data not shown). These results strongly supportedthe possibility that NMU, by interaction with GHSR1b and NTSR1, mightplay a very significant role in development/progression of NSCLC.

Identification of Downstream Genes of NMU

To further elucidate the NMU-signaling pathway and identify downstreamgenes regulated by NMU, siRNA against NMU (si-NMU) or LUC (controlsiRNA) were transfected into LC319 cells which had overexpressed NMU anddown-regulations in gene expression were monitored using a cDNAmicroarray that contained 32,256 genes. Among hundreds of genes detectedby this method, we performed Self-organizing map (SOM) clusteringanalysis to further select candidate genes. SOM clustering is datamining and visualization method originally developed by Kohonen(Kohonen, T. (1990). The self-organizing map. IEEE 78, 1464-1480.) andapplied to the analysis of gene expression data from microarrays. Theclustering method is similar to k-means clustering (Kaech, S. M., etal., (2002). Cell 111, 837-851.) but differs in that genes are dividedinto groups based on expression patterns, and relationships betweengroups are illustrated by two-dimensional maps. The genes passing ourvariation filter were grouped by a 5×4 SOM.

We initially selected 70 genes using SOM cluster analysis, whoseintensity were significantly decreased in accordance with the reductionof NMU expression (FIG. 15 a). Semiquantitative RT-PCR analysisconfirmed reduction of candidate transcripts in a time-dependent mannerin LC319 cells transfected with si-NMU, but not with control siRNA forLUC (FIG. 15 b). These transcripts were also confirmed to beup-regulated greater than 2-fold in LC319 cells expressing exogenousNMU, compared with that of normal lung tissues. Overexpression of thesegenes in accordance with NMU expression were evaluated as well inlung-cancer tissues and cell lines (data not shown). We finallyidentified 6 candidate NMU target genes, which satisfied the aboveselection criteria; FOXM1, FLJ42024, GCDH, CDK5RAP1, LOC134145, andNUP188 (FIG. 15 b).

FOXM1 mRNA levels were significantly elevated in lung cancers comparedwith normal lung tissues and its expression showed good concordance withNMU and two receptors for NMU, GHSR1b and NTSR1, whereas the function ofFOXM1 in lung carcinogenesis remains unclear. Therefore, we chose FOXM1for further analysis. To determine specific induction of the FOXM1 bythe NMU ligand-receptor signaling, LC319 cells expressing GHSR1b andNTSR1 were cultured in the presence of NMU-25 or BSA (control) at finalconcentrations of 100 μM in the culture media. NMU-25-treated cellsshowed higher expression of FOXM1 compared to the control cells (FIG. 15c). Furthermore, FOXM1 was also confirmed to be up-regulated in LC319cells expressing exogenous NMU, compared with that of control cellstransfected with mock vector (data not shown).

We then examined the biological significance of the FOXM1 activation byNMU signaling for growth or survival of lung-cancer cells, usingplasmids designed to express siRNA against FOXM1 (si-FOXM1).Transfection of si-FOXM1 into A549 or LC319 cells suppressed expressionof the endogenous FOXM1 in comparison to cells containing any of thethree control siRNAs (FIGS. 16 a and 16 b). In accordance with thereduced expression of the FOXM1, A549 and LC319 cells showed significantdecreases in cell viability and numbers of colonies (FIG. 16 a and b).These results strongly demonstrated that NMU, by the interaction withGHSR1b/NTSR1 and subsequent activation of its downstream targets, suchas FOXM1, could significantly affect the growth of lung-cancer cells.

Microarray data of LC319 cells treated with siRNA for NMU presentedherein proved that NMU signaling pathway could affect the growthpromotion of lung-cancer cells by transactivating a set of downstreamgenes involving transcripts whose protein products can function as atranscription factor and are capable of controlling cell growth orparticipating in signal transduction. We provided evidence that theFOXM1 transcription factor is a downstream target of NMU signaling byadditional biological assays. FOXM1 was known to be over-expressed inseveral types of human cancers (Teh, M. T. et al., Cancer Res. 62,4773-4780; van den Boom, J. et al., (2003). Am. J. Pathol. 163,1033-1043; Kalinichenko, V. V. et al., (2004). Genes. Dev. 18, 830-850).The “forkhead” gene family, originally identified in Drosophila,comprises transcription factors with a conserved 100-amino acidDNA-binding motif, and has been shown to play important roles inregulating the expression of genes involved in cell growth,proliferation, differentiation, longevity, and transformation.Cotransfection assays in the human hepatoma HepG2 cell line demonstratedthat FOXM1 protein stimulated expression of both the cyclin B1 (CCNB1)and cyclin D1 (CCND1) (Wang, X. et al., (2002). Proc. Nat. Acad. Sci.99, 16881-16886.), suggesting that these cyclin genes are direct FOXM1transcription targets and that FOXM1 controls the transcription networkof genes that are essential for cell division and exit from mitosis. Itshould be noted that we observed activation of CCNB1 in the majority ofa series of NSCLC we examined and its good concordance of the expressionto FOXM1 (data not shown). On the other hand, it was also demonstratedthat p27 (Kip1) and p19 (Arf) (CDKN2A) interact with FOXM1 and inhibitFOXM1 transcriptional activity (Kalinichenko, V. V. et al., (2004).Genes. Dev. 18, 830-850). The promotion of cell growth in NSCLC cells byNMU might reflect transactivation of FOXM1, which would affect thefunction of those molecular pathways in consequence.

By immunohistochemical analysis on tissue microarray, we detectedincreased expression of NMU protein in the majority of NSCLC (SCC, ADC,LCC, and BAC) and SCLC samples, but not in normal lung tissues. SinceNMU is a secreted protein and most of the clinical NSCLC samples usedfor our analysis were at an early and operable stage, NMU might serve asa biomarker for diagnosis of early-stage lung cancer, in combinationwith fiberscopic transbronchial biopsy (TBB) or blood tests.

In summary, we have shown that NMU and two newly revealed receptors forthis molecule, GHSR1b and NTSR1, are likely to play an essential rolefor an autocrine growth-promoting pathway in NSCLCs by modulatingtranscription of down stream target genes. The data reported herestrongly imply the possibility of designing new anti-cancer drugs,specific for lung cancer, that target the NMU-GHSR1b/NTSR1 pathway. Theyalso suggest a potential for siRNAs themselves to interfere with thispathway, as a novel approach to treatment of chemotherapy-resistant,advanced lung cancers.

INDUSTRIAL APPLICABILITY

The expression of human genes KIF11, GHSR1b, NTSR1 and FOXM1 aremarkedly elevated in non-small cell lung cancer (NSCLC) as compared tonormal lung tissues. Accordingly, these genes can be conveniently usedas diagnostic markers of NSCLC and the proteins encoded thereby may beused in diagnostic assays of NSCLC.

The present inventors have also shown that the expression of KIF11,GHSR1b, NTSR1 or FOXM1 promotes cell growth whereas cell growth issuppressed by small interfering RNAs corresponding to KIF11, GHSR1b,NTSR1 or FOXM1 gene. These findings show that each of KIF11, KOC1,GHSR1b, NTSR1 and FOXM1 proteins stimulate oncogenic activity. Thus,each of these oncoproteins is a useful target for the development ofanti-cancer pharmaceuticals. For example, agents that block theexpression of KIF11, KOC1, GHSR1b, NTSR1 or FOXM1, or prevent itsactivity may find therapeutic utility as anti-cancer agents,particularly anti-cancer agents for the treatment of NSCLC. Examples ofsuch agents include antisense oligonucleotides, small interfering RNAs,and ribozymes against the KIF11, KOC1, GHSR1b, NTSR1 or FOXM1 gene, andantibodies that recognize KIF11, KOC1, GHSR1b, NTSR1 or FOXM1polypeptide.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method of screening for a compound for treating or preventingNSCLC, said method comprising the steps of: contacting a KIF11polypeptide or functional equivalent thereof with KOC1 polypeptide orfunctional equivalent thereof in the presence of a test compound;detecting the binding between the polypeptides; and selecting the testcompound that inhibits the binding between the polypeptides.
 2. Themethod of claim 1, wherein the functional equivalent of KIF11polypeptide comprises amino acid sequence of KOC1 binding domain.
 3. Themethod of claim 1, wherein the functional equivalent of KOC1 polypeptidecomprises amino acid sequence of KIF11 binding domain.